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Nucleic Acids Research , 2024, 1–23
https://doi.org/10.1093/nar/gkad1221
RNA and RNA-protein complexes
Het erog eneous nuclear ribonucleoprotein K promotes
cap-independent translation initiation of retroviral mRNAs
Yazmín F uent es
1 ,†
, Val eria Olguín
1 ,†
, Brenda López-Ulloa
1
, Dafne Mendonça
1
, Hade Ramos
1
,
Ana Luiza Abdalla
2 , 3
, Gabriel Guajar do-Contrer as
2 , 4
, Meijuan Niu
2
, Barbar a Rojas-Ar a y a
1
,
Andr ew J . Mouland
2 , 3 , 4 and Marcelo López-Lastra
1 ,
*
1
Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e
Inmunología Pediátrica, Escuela de Medicina, Ponticia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile
2
HIV-1 RNA Trafcking Laboratory, Lady Davis Institute at the Jewish General Hospital, Montréal, Quebec H3T 1E2, Canada
3
Department of Microbiology and Immunology, McGill University, Montreal, Quebec H4A 3J1, Canada
4
Department of Medicine, McGill University, Montreal, Quebec H4A 3J1, Canada
*
To whom correspondence should be addressed. Te l: +56 22 354 3410; Email: malopez@med.puc.cl
†
The rst two authors should be regarded as Joint First Authors.
Abstract
Translation initiation of the human immunodeciency virus-type 1 ( HIV-1 ) genomic mRNA ( vRNA ) is cap-dependent or mediated by an internal
ribosome entry site ( IRES ) . The HIV-1 IRES requires IRES-transacting f actors ( I TAFs ) f or function. In this study, w e e v aluated the role of the
heterogeneous nuclear ribonucleoprotein K ( hnRNPK ) as a potential I TA F f or the HIV-1 IRES. In HIV-1-expressing cells, the depletion of hnRNPK
reduced HIV-1 vRNA translation. Furthermore, both the depletion and o v ere xpression of hnRNPK modulated HIV-1 IRES activity. Phosphorylations
and protein arginine methyltransferase 1 ( PRMT1 ) -induced asymmetrical dimethylation ( aDMA ) of hnRNPK strongly impacted the protein’s ability
to promote the activity of the HIV-1 IRES. We also show that hnRNPK acts as an I TA F f or the human T cell lymphotropic virus-type 1 ( HT LV-
1 ) IRES, present in the 5
UTR of the viral sense mRNA, but not for the IRES present in the antisense spliced transcript encoding the HTLV-1
basic leucine zipper protein ( sHBZ ) . This study provides evidence for a novel role of the host hnRNPK as an ITAF that stimulates IRES-mediated
translation initiation for the retroviruses HIV-1 and HTLV-1.
Gr aphical abstr act
IRES-mediated Translation
hnRNPK
n
dl HIV-1 IRES
dl HTLV-1 IRES +hnRNPK
RLUC ΔEMCV HIV-1 IRES FLUC
Cap AAA(A)n
RLUC ΔEMCV HTLV-1 IRES FLUC
Cap AAA(A)n
hnRNPK
IRES-mediated Translation
PRMT1
M
5RK
5RG
P
P
P
P
P
P
P
S284 S353
P
S216
Y458
dl HIV-1 IRES + (PTM)-hnRNPK
P
P
RLUC ΔEMCV HIV-1 IRES FLUC
Cap AAA(A)n
Introduction
The human immunodeciency virus-type 1 ( HIV-1 ) , the
causative agent of the acquired immune deciency syndrome
( AIDS ) pandemic, elicits multiple strategies to guarantee the
initiation of viral mRNA ( vRNA ) translation ( 1 ) . These strate-
gies ensure the synthesis of the structural polyproteins Gag
and Gag / Pol, required for progeny virus production. Transla-
tion initiation of the HIV-1 vRNA occurs via canonical and
non-canonical cap-dependent mechanisms or by using an in-
ternal ribosome entry site ( IRES ) -dependent mechanism (
1–
3 ) . The HIV-1 vRNA harbors two IRESs, the HIV-1 IRES
( the main focus of this study ) within the 5
untranslated re-
gion ( UTR ) and the Gag-IRES within the gag -coding region
( 4–6 ) .
In cells, translational control is primarily exerted during
the initiation step of protein synthesis, a multistep process
that leads to the assembly of the 80S ribosome at the start
codon of the mRNA ( 7 ) . The rst step in canonical translation
Received: April 25, 2023. Revised: December 7, 2023. Editorial Decision: December 10, 2023. Accepted: December 15, 2023
©The Author ( s ) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http: // creativecommons.org / licenses / by / 4.0 / ) ,
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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2 Nucleic Acids Research , 2024
initiation involves 5
cap ( m7GpppN ) recognition by the eu-
karyotic initiation factor ( eIF ) 4F, a heterotrimeric complex
comprising the cap-binding protein, eIF4E, an ATP-dependent
RNA helicase, eIF4A, and the scaffold protein, eIF4G ( 7 ) .
Also, eIF4G bridges the interaction with the 40S ribosomal
subunit via eIF3 and interacts with the poly ( A ) -binding pro-
tein ( PABP ) that covers the poly ( A ) tail, stimulating transla-
tion initiation, translation reinitiation through ribosome re-
cycling and enhancing mRNA stability . HIV -1 targets cap-
dependent translation initiation during replication ( 1 ,2 ) such
that in HIV-1 replicating cells, eIF4E and the eIF4E-binding
protein are hypophosphorylated, reducing cap-dependent
translation initiation ( 8 ) . Also, the HIV-1 protease cleaves
eIF4G and PABP, impeding eIF4E-eIF4G and eIF4G-PABP
interactions, negatively impacting cap-dependent translation
initiation ( 9 ) . Furthermore, HIV-1 replication induces oxida-
tive and osmotic stress and blocks the cell cycle in G2 / M,
physiological conditions in which cap-dependent translation
initiation is hindered ( 8 ,10–12 ) . While HIV-1 inhibits cap-
dependent translation initiation, non-canonical translation
initiation sustains Gag polyprotein synthesis ( 4 , 13 , 14 ) . The
HIV-1 IRES remains functional when cap-dependent transla-
tion initiation is repressed by drug-induced cell cycle block-
age in G2 / M ( 4 ) , by the expression of the poliovirus ( PV )
2A or the foot and mouth disease virus ( FMDV ) L proteases,
viral enzymes that cleave eIF4G, specically inhibiting cap-
dependent translation ( 4 , 13 , 14 ) , and in cells where HIV-1 and
poliovirus co-replicate enabling HIV-1 Gag protein synthesis
( 14 ) .
The molecular mechanisms driving HIV-1 IRES activity
are still poorly understood ( 1 ,2 ) . However, function of the
HIV-1 IRES relies on host proteins such as eIF4A and eIF5A
( 15–17 ) , the ribosomal protein S25 ( 18 ) , and on IRES trans-
acting factors ( ITAFs ) that activate or repress its activity
( 6 , 11 , 15 , 19–23 ) . The known ITAFs for the HIV-1 IRES in-
clude the heterogeneous nuclear ribonucleoprotein ( hnRNP )
A1 ( 11 ,21 ) , Staufen1 ( 22 ) , the human Rev-interacting protein
( hRIP ) ( 15 ) , DDX3 ( 15 ) , the Human antigen R ( HuR ) ( 19 ) ,
and upstream of N-ras ( unr ) ( 23 ) . Pulldown / mass spectrom-
etry experiments have identied additional proteins that bind
the 5
UTR of the HIV-1 vRNA ( 24 ) . Among the identied pro-
teins, several members of the hnRNP family of RNA-binding
proteins ( RBP ) , including hnRNPA1, hnRNPI, hnRNPK, hn-
RNPU, and hnRNPF, are found ( 24 ) . However, except for hn-
RNPA1 and hnRNPI ( 11 , 21 , 25 ) , the roles of other hnRNPs
identied as binding partners of the HIV-1 5
UTR in HIV-1
IRES-mediated translation initiation have not been evaluated.
From the hnRNPs identied to bind the HIV-1 vRNA 5
UTR
( 24 ) , we were interested in assessing the role of the ubiqui-
tously expressed, multifunctional nucleocytoplasmic shuttling
RBP, hnRNPK, on HIV-1 IRES activity because of its many
roles in HIV-1 gene expression ( 26–29 ) , but heretofore un-
resolved function in IRES-mediated vRNA translation initi-
ation. Consistent with this role in viral gene expression, the
knockdown of hnRNPK in cells actively replicating HIV-1
decreases intracellular viral proteins and reduces HIV-1 pro-
duction ( 28 ) . Other factors that pointed to hnRNPK as an
interesting target for this study was that the protein is an
ITAF for other viral and cellular IRESs ( 30–32 ) , and in cells,
it forms protein-protein complexes with known HIV-1 IRES
ITAFs such as hnRNPA1, HuR, and DDX3, and interacts with
other proteins required for HIV-1 IRES function, such as eS25
( 33 ,34 ) .
This study reveals that hnRNPK promotes HIV-1 IRES ac-
tivity. Our results show that post-translational modications
( PTMs ) of hnRNPK modulate the protein’s ability to stimu-
late HIV-1 IRES-mediated translation initiation. Our ndings
demonstrate that hnRNPK also promotes the activity of the
IRESs present within the 5
UTR of human T cell lymphotropic
virus-type 1 ( HTLV-1 ) vRNA, without having any impact on
the activity of the IRES present in the HTLV-1 antisense RNA,
encoding for the HTLV-1 basic leucine zipper ( HBZ ) protein.
Materials and methods
Plasmids
The pNL4.3 DNA ( HIV-1 vector; GenBank: AF324493 ) was
obtained through the NIH AIDS Reagent Program, Division
of AIDS, NIAID, NIH. The HIV-1 pNL-4.3-RLuc provirus
and the plasmid pEGFP-C1 were kindly provided by Dr. R.
Soto-Rifo ( Laboratorio de Virología Molecular y Celular, Pro-
grama de Virología, Instituto de Ciencias Biomédicas, Univer-
sidad de Chile, Santiago, Chile ) and were described in de-
tail ( 35 ) . The dual-luciferase ( dl ) plasmids dl HIV-1 IRES,
harboring the 5
UTR of the HIV-1 vRNA ( 1-336 ) , dl HIV-1
IRES 104–336, dl HIV-1 IRES 1–104, SV40 dl HIV-1 IRES,
dl HTLV-1 IRES, and dl sHBZ IRES were described ( 4 ,36–
38 ) . The hnRNPK plasmid ( pCMV-HA-hnRNPK ) was pur-
chased from Sino Biological Inc. ( #HG16029-NY, Wayne, PA,
USA ) . The selected hnRNPK substitutions have been previ-
ously described ( 39–44 ) . Mutants were generated using the
Thermo Fisher Scientic Phusion Site-Directed Mutagenesis
Kit ( #F-541, Thermo Fisher Scientic Inc. Life Technologies
Inc., Carlsbad, C A, US A ) using primers described in Tab l e 1 .
The polymerase chain reaction ( PCR ) assays were performed
in a Ver iti TM 96-well Thermal Cycler ( #4375768, Thermo
Fisher Scientic Inc. ) . All constructs used in this study were
veried by sequencing ( Psomagen Inc., Rockville, MD, USA ) .
The hnRNPK plasmids, GFP-hnRNPK and GFP-hnRNPK-
5RG, were generously provided by Dr. A. Ostareck-Lederer
( Department of Intensive Care Medicine, University Hospital
RWT H Aachen, Aachen, Germany ) and have been described
( 44 ) .
Cell culture and drug treatments
HeLa cells ( ATC C CCL-2 ) and human embryonic kidney
cells ( HEK293T, ATCC, CRL-11268 ) were grown as previ-
ously described in detail ( 21 ,22 ) . The PRMT1 inhibitor TC-
E 5003 ( CAS 17328-16-4, Santa Cruz Biotechnology, Dallas,
TX, USA ) was diluted in DMSO. HEK293T were seeded at
1.2 ×10
5 and 6 ×10
4 per well in a 24- or 48-well culture
plates, respectively. 24 h later, cells at 60% conuence were
treated with DMSO ( control ) or TC-E 5003 at the indicated
compound concentrations for 24 h.
DN A tr ansfection
HEK293T cells were seeded at 1.2 ×10
5
or 6 ×10
4
per well
in a 24- or 48-well culture plates, respectively. DNA trans-
fection experiments were performed at 70–80% conuency
using polyethyleneimine ( PEI; GIBCO, Thermo Fisher Scien-
tic Inc. ) . The cells were cotransfected with 200 ng of dl-
plasmids, together with the indicated amount ( ng ) of HA-
hnRNPK plasmids, GFP-hnRNPK plasmids or plasmid pSP64
Poly ( A ) ( #P1241, Promega Corporation, Madison, WI, USA ) ;
the last was used as a ller DNA to keep the nal DNA
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Nucleic Acids Research , 2024 3
Ta b l e 1. Primers used to generate the hnRNP K point mutants
Mutant Primers sequences
HNRNP K S284A Fw: 5
( P ) TGA TGA T A TGGCCCCTCGTCGA
Rv: 5
( P ) T AA TCTCTTCT AGA TGGAGGCA TGGG
HNRNP K S284D Fw: 5
( P ) TGA TGA T A TGGACCCTCGTCGA
Rv: 5
( P ) T AA TCTCTTCT AGA TGGAGGCA TGGG
HNRNP K S353A Fw: 5
( P ) AGA T ACA TGGGCCCCA TCAGAA
Rv: 5
( P ) ATTGCA GA GTCCCAA GTTTCATC
HNRNP K S353D Fw: 5
( P ) AGA T ACA TGGGACCCA TCAGAA
Rv: 5
( P ) ATTGCA GA GTCCCAA GTTTCATC
HNRNP K S216A Fw: 5
( P ) CTT A T A TCTGAGGCTCCCA T
CAAA GGA CGT
Rv: 5
( P ) A TCAAGGA TGA TCTTT A TGCA
CTCTA CAA C
HNRNP K S216D Fw: 5
( P ) CTT A T A TCTGAGGA TCCCA TC
AAA GGA CGT
Rv: 5
( P ) A TCAAGGA TGA TCTTT A TGCA
CTCTA CAA C
HNRNP K Y458A Fw: 5
( P ) A GTGTGAA GCA GGCTGCA
GATGTTGAA
Rv: 5
( P ) GTTCTGC AGCAAATACTGTGC ATTCT
GTATCTG
HNRNP K Y458D Fw: 5
( P ) A GTGTGAAGCA GGATGCAGATGTTGAA
Rv: 5
( P ) GTTCTGC AGCAAATACTGTGC ATTCT
GTATCTG
HNRNP K Y458F Fw: 5
( P ) A GTGTGAAGCA GTTTGCAGATGTTGAA
Rv: 5
( P ) GTTCTGC AGCAAATACTGTGC ATTCT
GTATCTG
HNRNP K Y458T Fw: 5
( P ) A GTGTGAAGCA GA CTGCAGATGTTGAA
Rv: 5
( P ) GTTCTGC AGCAAATACTGTGC ATTCT
GTATCTG
HNRNP K Y458S Fw: 5
( P ) A GTGTGAAGCA GA GTGCAGATGTTGAA
Rv: 5
( P ) GTTCTGC AGCAAATACTGTGC ATTCT
GTATCTG
HNRNP K Y458E Fw: 5
( P ) A GTGTGAA GCA GGA GGC
AGATGTTGAA
Rv: 5
( P ) GTTCTGC AGCAAATACTGTGC ATTCT
GTATCTG
HNRNP K Y458R Fw: 5
( P ) A GTGTGAAGCA GA GA GCAGATGTTGAA
Rv: 5
( P ) GTTCTGC AGCAAATACTGTGC ATTCT
GTATCTG
HNRNP K R256K Fw: 5
( P ) TTTCCCATGAA GGGAA GA GGTGGT
Rv: 5
( P ) TCCCACTGGGCGTCCGCG
HNRNP K R299K Fw: 5
( P ) GA GGCGGCAA GGGTGGTA GC
Rv: 5
( P ) GTCCGGGA GGA GGGGGA GG
concentration constant in all transfection assays. In all ex-
periments, 24 or 48 h post-transfection ( hpt ) , the culture
medium was removed, and cells were harvested using Passive
Lysis buffer supplied with the Dual-Luciferase® Reporter As-
say System ( #E1960, Promega Corporation ) according to the
manufacturer’s protocols.
siRN A-DN A co-tr ansfection
HEK293T cells were seeded at 1.2 ×10
5 cells per well
in 24-well culture plates. Endogenous hnRNPK protein si-
lencing was performed over 70% conuent cells using the
Lipofectamine 2000 system ( #11668019, Invitrogen, Thermo
Fisher Scientic Inc. ) . For hnRNPK silencing, a commer-
cially available mix of silencing RNAs that target the hn-
RNPK open reading frame ( ORF ) , siRNAK ( #sc-38282, Santa
Cruz Biotechnology ) , a combination of ve target-specic siR-
NAs against the hnRNPK ORF, or a Dicer-Substrate siRNA
( DsiRNA ) targeting the 3´UTR of the hnRNPK encoding
mRNA ( hs.Ri.HNRNPK.13.3, Integrated DNA Technolo-
gies, IDT, Coralville, IA, USA ) , DsiRNAK, were used. As
negative controls, a scrambled siRNA ( scRNA; #4390844;
Ambion, ThermoFisher Scientic Inc ) or scDsiRNA ( #51-
01-14-04, IDT ) . For RLuc silencing, 50 nM of a duplex
siRNA targeting the RLuc open reading frame ( siRLuc, 5
-
U AU AAGAACC AUU ACC AGAUUUGCCUG-3
, ( IDT ) ) was
used as described previously ( 18 , 21 , 22 , 38 ) .
Luciferase assays
The activities of rey luciferase ( FLuc ) and Renilla lu-
ciferase ( RLuc ) were measured using the DLR® Assay System
( #E1960, Promega Corporation ) according to the manufac-
turer’s instructions on 10 μl of cell lysates using a Sirius Sin-
gle Tube Luminometer ( Berthold Detection Systems, GmbH,
Pforzheim, Germany ) . Data are expressed as a percentage of
the relative luciferase activity ( RLA ) or as relative translation
activity ( R TA ) . The R TA corresponds to the FLuc / RLuc ratio
and is used as an index of the IRES activity ( 6 , 20 , 36 ) .
RN A extr action and real-time RT-qPCR
HEK293T cells were seeded at 2.4 ×10
5
cells per well in 12-
well culture plates, and cytoplasmic RNA was extracted. Cells
were washed twice with PBS 1 ×( #SH30256, Hyclone, GE
Healthcare Life Sciences, Boston, MA, USA ) , and incubated 5
min on ice with RNLa buffer ( 10 mM Tris – HCl pH 8, 10 mM
NaCl, 3 mM MgCl
2
, 0.5% NP-40, 1 mM DTT ) containing
10 U of RNase inhibitor ( #EO0381, Thermo-Fisher Scientic
Inc. ) . After incubation, 500 μl of TRIzol reagent ( #15596018,
Life Technologies Corporation, Thermo Fisher Scientic Inc. )
was added to the supernatant, and the RNA was recovered.
Cytoplasmic RNA was resuspended in 25 μl of nuclease-free
water, DNase-treated ( #AM1907, Ambion, Thermo-Fisher
Scientic Inc. ) , and recovered according to the manufacturer’s
instructions. The RNA concentration was quantied by nano-
spectrophotometry ( N60-Implen Nanophotometer, West l a ke
Village, C A, US A ) . The relative RNA quantication was car-
ried out by real-time reverse transcription ( RT ) -quantitative
polymerase chain reaction ( qPCR ) assay ( QuantStudio 3
Real-Time PCR System, Thermo Fisher Scientic Inc. ) us-
ing the Brilliant II SYBR Green RT-qPCR one Step Master
Mix ( #600835, Agilent Technologies, Santa Clara, C A, US A ) .
Gag-RLuc RNA was amplied using primers Renilla sense ( 5
-
A GGTGAA GTTCGTCGTCCAA CA TT A TC-3
) and Renilla
antisense ( 5
-GAAA CTTCTTGGCA CCTTCAA CAA T AGC-
3
) as previously described ( 18 ) . When required, to es-
tablish the amount of RLuc RNA, the Renilla sense
and Renilla antisense primers were used. To deter-
mine the amount of FLuc RNA, the FLuc sense ( 5
-
ACTTCGAAATGTCCGTTCGG-3
) and FLuc antisense
( 5
- GCAACTCCGA T AAA T AACGCG-3
) primers were used
( 38 ) . No-RT-qPCR reactions were carried out to control
for contaminant DNA Glyceraldehyde-3-phosphate dehy-
drogenase ( GAPDH ) mRNA was detected with the primers
GAPDH sense ( 5
-TCC ACC ACCCTGTTGCTGTAG-3
) and
GAPDH antisense ( 5
-A CCCA CTCCTCCA CCTTTGA C-3
)
( 45 ) . Data analysis was performed by the Ct method as
previously described in ( 46 ) .
Cell viability
The cell viability assay was performed using the CellTiter
96® Aqueous One Solution Cell Proliferation Assay ( MTS )
( #G358A, Promega Corporation ) according to the manufac-
turer’s instructions. Briey, HEK293T cells were seeded at
1.5 ×10
3 cells per well in a 96-well plate and transfected
with the indicated concentrations of hnRNPK ( pCMV3-HA
hnRNPK wt or mutants ) expressing plasmids or the PRMT1
inhibitor TC-E 5003, the CellTiter 96® Aqueous One Solu-
tion Cell Proliferation Assay was added, incubated at 37
◦C
for 4 h, and the absorbance was measured at 495 nm in a
Biochrom EZ Read 400 microplate reader ( Biochrom, Hollis-
ton, MA, USA ) .
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4 Nucleic Acids Research , 2024
Wes t ern blots
Cells were lysed using the Passive Lysis 5 ×Buffer ( #E1941,
Promega Corporation ) . The concentration of total protein
was determined by the Bradford assay using the Bio-Rad Pro-
tein Assay ( #5000006, Bio-Rad Laboratories, Inc., Hercules,
C A, US A ) . Equal amounts of protein ( 30 or 40 μg ) were re-
solved by electrophoresis on a 10% or 12% glycine sodium
dodecyl sulfate-polyacrylamide gel ( SDS-PAGE ) transferred
onto a 0.45 μm nitrocellulose membrane ( #10600002; GE
Healthcare Bio-Sciences 100 ResultsWay, Marlborough, MA,
USA ) . Membranes were blocked with Tris-buffered saline ( pH
7.4 ) containing 5% skimmed milk and 0.1% Tween-20 ( TBS-
T ) for 1 h at room temperature and incubated overnight at
4
◦C with the primary antibody. The membranes were washed
three times with TBS-T and incubated with the correspond-
ing horseradish peroxidase-conjugated secondary antibodies.
The primary mouse anti-hnRNPK antibody ( #sc-28380, Santa
Cruz Biotechnology ) was used at 1:500 dilution, a mouse anti-
DDX3 ( #ab50703, Abcam, Cambridge, UK ) , mouse anti-HuR
( sc-5261, Santa Cruz Biotechnology ) , and a rabbit anti-p17
HIV-1 ( #4811, NIH AIDS Reference and Reagents program )
were used at 1:1000 dilution, a mouse anti-HA ( #H9658,
Sigma-Aldrich, 3050 Spruce Street, St. Louis, MO, USA ) , a
mouse anti-GFP ( #632381, Living Colors® A.v. Monoclonal
Antibody JL-8, Clontech Laboratories, Inc., C A, US A ) , mouse
anti-PARP-1 ( sc-136208, Santa Cruz Biotechnology ) , mouse
anti-hnRNPA1 ( #sc-32301, Santa Cruz Biotechnology ) , and
a mouse anti-GAPDH ( #MA5-15738; Thermo Fisher Scien-
tic Inc. ) were used at a 1:5000 dilution. For stripping, mem-
branes were incubated with stripping buffer ( glycine 0.2 M,
NaCl 0.5 M pH 2.8 ) for 15 min at room temperature, washed
with TBS-T ( Tris-buffered saline ( pH7.4 ) 0.1% Tween-20 )
for 15 min at room temperature. The incubation and wash-
ing steps were repeated twice. Membranes were blocked with
TBS-T containing 5% skimmed milk for 1 h at room tempera-
ture, washed three times with TBS-T, and incubated overnight
at 4
◦C with the primary antibody. Either a Goat anti-mouse
or Goat anti-rabbit IgG-horseradish peroxidase ( HRP ) conju-
gate ( #AP308P, #AP132P; Merck, Darmstadt, Germany ) sec-
ondary antibodies, both at 1:10 000 dilution, were used. The
Asymmetric Di-Methyl Arginine ( aDMA ) Motif [adme-R]
MultiMab™ Rabbit mAb mix ( #13522, Cell Signaling Tech-
nology, Danvers, MA, USA ) used at a 1:1000 dilution. Wes t e rn
blots were visualized by enhanced luminescence by a chemilu-
minescence reaction using 4-hydroxycinnamic acid ( #800237,
Merck ) and luminol ( #09253, Sigma-Aldrich ) , the SuperSig-
nal™ West Femto Maximum Sensitivity Substrate ( #34096,
Thermo Fisher Scientic Inc. ) or We s tern Lightning Plus-ECL
( # NEL 121001, PerkinElmer Health Science Canada, Inc, On-
tario, Canada ) . The western blot lms ( Fuji medical X-ray lm
Super HR-U 30 ) or Hyblot CL ( # DV-3012, Denville Scientic
Inc., NJ, USA ) ) were digitized using a CanonScan 9950F scan-
ner or captured using an Alliance 2.7 imaging system ( UVItec
Cambridge, Topac Inc., 231 CJC Highway, Cohasset, MA,
USA ) ( 21 ,22 ) .
Co-immunoprecipitation ( CoIP )
For the CoIP assays, 3 ×10
6
HEK 293T cells were transfected
with the dl HIV-1 IRES plasmid together with pCMV-HA-
hnRNPK or pCMV-HA-hnRNPK-5RK plasmids. 48 hpt, cells
were washed three times with PBS 1X and resuspended in lysis
buffer ( NaCl 100 mM, EDTA 2mM, Tris – HCl 50 mM pH 7.5,
Naf 50 mM, sodium orthovanadate 1 mM, Trit o n X-100 1%,
and protease inhibitors ( #11836170001, Roche Diagnostics,
Sigma-Aldric ) . The protein concentration in lysates was deter-
mined by Pierce BCA Protein assay ( #23227, Thermo Fisher
Scientic Inc ) . Protein A / G Plus-Agarose ( #SC-2003, Santa
Cruz Biotechnology ) beads were incubated with the anti-HA
mouse ( #H9658, Sigma-Aldrich ) or anti-IgG mouse antibody
( #SC-2025, Santa Cruz Biotechnology ) for 4 h. Beads were
washed once with lysis buffer to discard unbound antibodies.
Then 0.5 mg of total protein was incubated with the antibody-
coated beads for 16 h at 4
◦C in rotation. The beads were
washed with lysis buffer three times, glycine loading buffer
( 5 ×) was added, and the mix was heated ( 95
◦C for 5 min ) .
The supernatant was recovered by centrifugation and sub-
jected to western blot assay ( as described above ) using protein
A / G conjugated with HRP ( # 32490, Thermo Fisher Scientic
Inc. ) as the secondary antibody.
Immunouorescence and microscopy
HeLa cells were seeded at 7 ×10
4 cells per well on a 12-
well plate previously prepared with sterilized cover glasses
of 0.15 mm thickness ( #16004-300, V .W .R. V istaV ision ) . 24
h later, cells were transfected with the HIV-1-coding plasmid
pNL4.3 or the empty vector pcDNA3.1, using JetPRIME®
( #101000027, Polyplus, Illkirch, France ) according to the
manufacturer’s recommendations. 24- or 48 hpt, coverslips
were washed twice with 1X D-PBS and xed in 4% PFA
for 15 min, followed by 0.1M glycine for 10 min at room
temperature. Cover glasses were blocked in a 1% BSA so-
lution diluted in 1X D-PBS for 45 min at room tempera-
ture. For permeabilization, coverslips were incubated in a
0.2% Tri ton X-100 solution in 1 ×D-PBS for 5 min and
washed before blocking. For mild permeabilization, 0.025%
Saponin was added to the blocking buffer and kept during
blocking and antibody incubation. After blocking, cells on
cover glasses were incubated for 1 h at 37
◦C with a cock-
tail of primary antibodies: rabbit anti-p24 ( #ARP-4250, NIH
HIV reagent program ) and mouse anti-hnRNPK ( #Ab39975,
Abcam ) or mouse anti-hnRNPA2 ( hybridoma EF67 ( 47 ) , gen-
erously provided by Dr. William Rigby, Dartmouth Med-
ical School, NH USA ) , all used at 1:100 dilution. Cover
glasses were washed four times in 1 ×D-PBS for 5 min and
incubated as before with a cocktail of secondary antibod-
ies ( Invitrogen donkey anti-rabbit-AlexaFluor 488 [A21206],
and donkey anti-mouse-AlexaFluor 594 [A21203], each used
at 1:300 dilution ) . The washing protocol was repeated, and
cover glasses were incubated with DAPI ( 4’,6-Diamidino-
2-Phenylindole, Dihydrochloride ) ( #D1306, Invitrogen ) , for
10 min. Cover glasses were dried at room temperature and
then mounted using a drop of Immu-Mount ( #9990402,
Thermo Fisher Scientic Inc ) . Images were acquired using the
Zeiss LSM800 confocal microscope using immersion oil in
a 40 ×/ 1.4 numerical aperture objective ) . Images were pro-
cessed and analyzed in Imaris v10.0.0 ( Bitplane, Andor Inc.,
Oxford Instruments, UK ) .
HEK293T cells were seeded at 2.5 ×10
4 cells per well
in a 24-well culture plate at 60% conuence, previously pre-
pared with sterilized cover glasses of 0.12 mm thickness, then
transfected with 200 ng of the pSP64 poly ( A ) , pCMV-HA-
hnRNPK or pNL4.3-Rluc plasmids. 48- or 72-hpt, depend-
ing on the treatment, the cells were washed with PBS 1 ×and
xed with 4% paraformaldehyde ( PFA ) for 10 min. Then,
the cells were permeabilized with PBS-Tr ( PBS 1X, Trit on
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Nucleic Acids Research , 2024 5
X-100 0.03% ) and blocked with 10% BSA in PBS-Tr for 1 h.
Cover glasses was incubated with primary antibodies in 5%
BSA in PBS-Tr overnight at 4˚C 16 h, mouse anti-hnRNP K
antibody ( sc-28380 ) was used at dilution 1:100, rabbit anti-
HA ( #H6908; Sigma-Aldrich ) , goat anti-eIF3 ( sc-16377 ) , and
rabbit anti-p24 were used at dilution 1:300. 24 h later the
cover glasses were washed 5 times with PBS-Tr and incubated
at room temperature for 2 h with a cocktail of secondary
antibodies ( Invitrogen; donkey anti-rabbit-AlexaFluor 647
[A31573] and donkey anti-mouse-AlexaFluor 488 [A21202],
each diluted 1:300 ) . The cover glasses were washed 3 times
with PBS-Tr, once with PBS, and once with ultrapure wa-
ter and incubated with Vectashield H1200 ( Vector Laborato-
ries, Inc, Burlingame, CA 94010, USA ) with 4,6-diamidino-
2-phenylindole ( DAPI ) as a mounting medium, sealed with
clear nail polish and stored at 4
◦C. The images were captured
with 40 ×or 63 ×magnication using a ZEISS microscope,
axio observer D.1 model, and processed with the ImageJ
program.
UV-CLIP
HEK 293T cells ( 9.0 ×10
6
) were transfected with the dl
HTLV-1 IRES, dl sHBZ IRES or dl EMCV IRES, and the
HA-hnRNP K encoding vector. 24 hpt cells were washed, cov-
ered with cold PBS 1X ( 7mL / 10 cm plate ) ( #SH30246.01,
HyClone ) , and UV-irradiated ( UV-254 nm, 400 mJ / cm
2
)
on ice in a UV chamber ( UVP, UV Crosslinker CL-1000,
Upland, C A, US A ) . Cells were scraped, collected by cen-
trifugation at 4ºC, and lysed using RIPA buffer ( Tris-HCl
pH 7.5 10mM, EDTA 1mM, NaCl 150mM, NP-40 0.5%,
Sodium deoxycholate 0.5%, SDS 0.1%, and protease in-
hibitors ( Roche Diagnostics ) , 3 μL Riboblock ( #EO0381,
Thermo Fisher Scientic Inc ) or SUPERase •In™ ( #AM2694,
Ambion, Life Technologies ) and 6 μl DNAseRQ1 ( #M6101,
Promega Corporation ) , and sonicated ( Sonic Ruptor 250,
Omini International The homogenizer Company , Kennesaw ,
GA, USA ) on ice. The total lysate was precleared with protein
A / G coated beads ( #SC-2003, Santa Cruz Biotechnology ) for
1 h in rotation at 4
◦C and centrifugated. The protein concen-
tration of the supernatant was determined using the Pierce™
B.S.A. assay ( #23227,Thermo Fisher Scientic Inc ) . 300 μl of
cell lysate was saved as input for RNA and protein extrac-
tion, 800 μl ( 1.5 mg of total proteins ) was allocated for RNA
extraction, and 500 μl ( 1 mg total protein ) of the lysate was
used for western blots. For immunoprecipitation assay ( IP ) ,
protein extracts were mixed with protein A / G beads loaded
with an anti-HA monoclonal antibody. After 16 h at 4ºC,
with rotation. For samples designated to western blots, the
beads were mixed with tricine loading buffer ( 2X ) and heated
( 90ºC for 10 min ) , loaded and resolved in a tris-tricine gel, and
transferred to a 0.45 μm PVDF membrane ( #88518, Thermo
Scientic Inc ) , the anti-HA primary antibody was used. The
recombinant protein A / G conjugated with HRP was used
as the secondary antibody ( #32490, Thermo Fisher Scientic
Inc ) . For RNA extraction, the beads were washed with Buffer
PK ( 0.05M Tris-HCl pH 8, 0.5M LiCl, 0.03M EDTA, 0.5%
SDS, 0.1% ß-mercaptoethanol and Proteinase K ( Invitrogen ) )
and with Buffer PK + urea 7 M. Then 500 μl of TRIzol
( #15596018, Life Technologies Corporation ) was added, and
RNA extraction proceeded as suggested by the manufac-
turer. The total RNA concentration was quantied using a
nano-spectrophotometer ( N60-Implen, Nanophotometer, CA,
US A ) . R T-qPCR assays to detect FLuc and GAPDH mRNA
were performed as described above. Fold Enrichment ( FE ) of
FLuc encoding RNA was calculated as 2
( −Ct [HA / IgG] )
as de-
tailed in ( 46 ) .
Subcellular fractionation
HEK293T cells ( 2.5 ×10
6
) were seeded in 90 mm culture
plate, and cells at 70–80% conuence were transfected with
4.2 μg of plasmids pCMV3-HA-HNRNPK or pSP64 Poly ( A )
according to the assay. 24 hpt, the culture medium was re-
moved, and the cells were resuspended in 1 ml of PBS 1 ×, then
centrifuged at 1000 ×g for 3 min at 4ºC. The pellet was resus-
pended in 1 ml of PBS 1 ×; 0.3 ml was used for the preparation
of the complete cell extract, and 0.7 ml was used to obtain the
nuclear and cytoplasmic fractions. For the preparation of the
complete cell extract, the cells were centrifuged at 1000 ×g
for 5 min at 4
◦C. The pellet was resuspended in 300 μl of RIPA
buffer ( Tri s –HCl pH 7.5 10 mM, EDTA 1mM, NaCl 150 mM,
NP-40 0.5%, sodium deoxycholate 0.5%, SDS 0.1% and pro-
tease inhibitor ( Roche Diagnostics ) ) and then sonicated on ice
at 40% amplitude for 20 s. The total cell extracts were stored
at –20ºC. Cells were centrifuged at 1000 ×g for 5 min at
4
◦C to obtain the nuclear and cytoplasmic fractions. Then,
the pellet was resuspended in 300 μl of buffer RLNa ( 10 mM
of Tris– HCl ( pH 8 ) , 10 mM NaCl, 3 mM MgCl2, 1 mM DTT,
0.5% NP40, 10 U / μl of RNAse inhibitor ( Thermo-Fisher Sci-
entic Inc. ) ) and incubated on ice for 5 min. The lysate was
centrifuged at 16 000 ×g for 3 min. The supernatant obtained
was recovered, centrifuged under the abovementioned condi-
tions, and preserved as the cytoplasmic fraction. The pellet
obtained was resuspended in 300 μl buffer RLNa and cen-
trifuged at 16 000 ×g for 3 min. The pellet was resuspended in
300 μl of RIPA buffer and sonicated on ice, and the lysate ob-
tained was preserved as the nuclear fraction. The specicity of
the preparation of the previously described extracts was eval-
uated by western blot assays, using anti-poly ( ADP-ribose )
polymerase ( PARP ) ( nuclear ) or anti-GAPDH ( cytoplasmic )
antibodies.
Statistical analysis
Graphics and statistical analysis were performed using the
Prism v9.5.1 software (GraphPad Software LLC, San Diego,
C A, US A), completing the statistical test indicated in the text
and gure legends.
Results
HnRNPK promotes HIV-1 Gag protein synthesis
A proteomic screening assay using HeLa extracts as the source
of proteins identied hnRNPK as a protein that interacts
with the 5
UTR of the HIV-1 vRNA (clone NL4.3) ( 24 ),
the region that harbors the HIV-1 IRES ( 4 ). We wondered
whether hnRNPK was a regulator of HIV-1 protein synthe-
sis. To address this question, we decided to use HEK293T
cells, which, together with HeLa cells, are common tools used
by us and others to study the molecular biology of HIV-1
( 4 , 9 , 11 , 14 , 21 , 22 , 24 ). HEK293T cells were cotransfected with
the HIV-1 clone NL-4.3-RLuc DNA together with a commer-
cial Dicer-Substrate siRNA (DsiRNA), targeting the 3´UTR
of the endogenous hnRNPK mRNA (DsiRNAK, 10nM), or
with a non-related scrambled control (DscRNA, 10nM). The
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6 Nucleic Acids Research , 2024
A
BCD
Figure 1. HnRNPK regulates HIV-1 Gag expression. ( A ) Schematic representation of the complete HIV-1 molecular clone pNL-4.3-RLuc. ( B ) HEK 293T
cells were cotransfected with the pNL-4.3-RLuc (200 ng) plasmid and 10 nM of a DsiRNA targeting the hnRNPK mRNA (DsiRNAK) or with a scrambled
RNA ((DscRNA); 10 nM) as a control. The expression of the HIV-1 Gag-RLuc-HA fusion protein and endogenous hnRNPK was determined 24 hpt by
western blot using the GAPDH protein as a loading control. Fo r the semi-quantitative comparative analysis, the captured images we re quantied using
the ImageJ 1. 5 3 software (Windows version of NIH ImageJ, http:// imagej.nih.gov/ ij ). Values expressed in percentages (%) correspond to the ratio (OD
value (p17 or hnRNPK) / OD value GAPDH) relative to the control (DscRNA) set to 100%. ( C ) Renilla luciferase activity was measured 24 hpt and is
expressed as relative luciferase activity (RLA) relative to the activity obtained when the pNL-4.3-RLuc plasmid was cotransfected with the DscRNA( −)
set to 100%. ( D ) Cytoplasmic RNA was extracted from cells expressing pNL-4.3-RLuc HIV-1 and treated with the DscRNA or the DsiRNAK (10 nM), and
relative RNA levels were determined by real-time RT-qPCR. The RNA abundance was expressed relative to the value obtained for the cells treated with
the DscRNA set to 10 0 % . In ( C ) and ( D ) , values represent the mean ( ±SEM) from six independent experiments, with each conducted in duplicate.
Statistical analyses wer e performed by an unpaired t wo-t ailed t -test (ns = nonsignicant; ** P ≤0.01).
pNL-4.3-RLuc plasmid (Figure 1 A) has a hemagglutinin
(HA)-ta gged Renilla luciferase (RLuc-HA) reporter gene in-
serted in frame with the group-specic antigen (Gag)-protein
start codon, generating a Gag-RLuc-HA fusion protein ( 35 ).
Transfecting the HIV-1 DNA enables us to skip the highly
regulated initiation steps in virus replication, including re-
ceptor recognition, entry, reverse transcription, and integra-
tion. Thus, we only focus on replication steps starting with
viral RNA transcription. As expected, treating cells with DsiR-
NAK decreased the detectable levels of endogenous hnRNPK
protein (Figure 1 B). In agreement with a previous report
( 28 ), the decrease of endogenous hnRNPK levels reduced
the amount of HIV-1 Gag-RLuc-HA protein, detected by us-
ing an antibody against Gag (anti-p17) (Figure 1 B). Consis-
tent with lower viral protein levels (Figure 1 B), a signicant
( P < 0.05) decrease in RLuc activity ( ∼43% reduction) in
cells treated with the DsiRNAK was also evidenced (Figure
1 C). Cytoplasmic RNA was extracted from pNL-4.3-RLuc
(DNA), DscRNA, or DsiRNAK cotransfected cells and used
as a template for quantitative analysis of the NL-4.3RLuc
RNA by RT-qPCR. The relative levels of NL-4.3-RLuc vRNA
were equivalent in the DscRNA- and DsiRNAK-treated cells
(Figure 1 D). Thus, the decrease in Gag-RLuc-HA levels in
HEK293T cells treated with the DsiRNAK RNA (Figure 1 B
and C) was not associated with a reduction in the abundance
of HIV-1 vRNA (Figure 1 D). These results indicate that a de-
cline in endogenous hnRNPK reduces HIV-1 vRNA transla-
tion, suggesting that hnRNPK plays a role in HIV-1 protein
synthesis.
HIV-1 gene expression does not induce a shift of
hnRNPK subcellular distribution
HnRNPK is a nucleo-cytoplasmic shuttling protein mainly
concentrated in the nucleus ( 48 ). HIV-1 replication does not
upregulate hnRNPK expression levels in infected cells ( 49 ).
However, HIV-1 replication may alter the protein’s subcellu-
lar distribution, as evidenced for hnRNPA2 ( 50 ). Thus, we
wondered whether HIV-1 gene expression induces a shift in
endogenous hnRNPK cellular compartmentalization. There-
fore, HeLa cells were transfected with pNL-4.3 DNA or con-
trol vector DNA, pcDNA3.1, and hnRNPK localization was
analyzed by indirect immunouorescence (IF). HIV-1 expres-
sion was monitored by detecting Gag protein in cells at 24
and 48 hpt along with endogenous hnRNPK (Figure 2 A and
C) or hnRNPA2 as a positive control (Figure 2 B and D). Be-
cause of the nuclear abundance of hnRNPK, cells were per-
meabilized using a strong (Triton X-100) or a mild (saponin)
agent, as Triton X-100 enhances signals from nuclear stain-
ing, while saponin enhances the cytoplasmic uorescence sig-
nal ( 51 ,52 ). As expected for a nucleo-cytoplasmic shuttling
protein, in HeLa cells, hnRNPK was detected in both the cell
cytoplasm (saponin) and the nuclear (Triton X-100) compart-
ments (Figure 2 A and C). In agreement with an earlier re-
port ( 48 ), hnRNPK localization was mainly nuclear. At 24
or 48 hpt of the pNL4.3 DNA, hnRNPK, and non-signicant
increasing trend of cytoplasmic hnRNPK was observed, but
most remained nuclear while HIV-1 Gag protein was cyto-
plasmic (Figure 2 A, C, and E). Under similar experimental
conditions, a fraction of hnRNPA2 was repositioned to the
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Nucleic Acids Research , 2024 7
F
E
CD
B
A
Figure 2. HIV-1 gene expression does not induce hnRNPK localization from the nucleus to the cytoplasm. HeLa cells were transfected with pNL4.3 or
the control vec tor pcDNA3.1. 24- and 48-hpt, cells were xed and permeabilized using Tri to n X-100 (strong detergent) ( A, B ) or saponin (mild detergent)
( C, D ). HeLa cells were stained against HIV-1 Gag (green) and hnRNPK (red) ( A, C ) or hnRNPA2 (red) ( B, D ). An o v ere xposed closer magnication on the
red channel, hnRNPK ( A, C ) or hnRNPA2 ( B, D ), is shown in the right column (Inset), where gray outlines the plasma membrane and blue the nucleus.
Scale bar = 15 μm (Inset = 5 μm). ( E, F ) hnRNPK and hnRNPA2 we re quantied in saponin-permeabilized cells by determining the mean uorescence
intensity (MFI) ratio between the cytoplasmic MFI and the overall cell MFI in the hnRNPK or hnRNPA2 channel. Individual cell values were obtained by
using the cell detection function of the Imaris v10.0 software. Each bar represents the mean value ( ±SEM) from 20 to 30 cells per condition. Statistical
analy ses w ere perf ormed b y multiple t -tests betw een the control and the HIV-1 condition per group. Statistical signicance w as determined using the
Holm–Sidak method (**** P ≤0.0 0 01).
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8 Nucleic Acids Research , 2024
cytoplasm (Figure 2 B, D and F) ( 50 ). Similarly, in HIV-1
expressing HEK293T cells, endogenous hnRNPK remained
mainly nuclear ( Supplemental Figure S1 ). Thus, we conclude
that in HeLa and HEK293T cells, HIV-1 gene expression does
not induce a detectable redistribution of endogenous hnRNPK
to the cell cytoplasm.
HnRNPK participates in HIV-1 IRES-mediated
translation in cells
Next, we assessed whether hnRNPK acted as an ITAF for
the HIV-1 IRES. Based on our earlier studies ( 4 , 18 , 20 , 21 , 36 ),
we used the well-characterized dual-luciferase (dl) reporter
plasmid dl HIV-1 IRES that encodes for a bicistronic mRNA
with an upstream Renilla luciferase (RLuc) ORF and a down-
stream rey luciferase (FLuc) ORF (Figure 3 A) ( 4 ). Placed
between both cistrons, the dl HIV-1 IRES RNA has a deleted
5
UTR of the encephalomyocarditis virus ( EMCV) RNA, a
highly structured element decient in IRES activity that im-
pedes ribosome reinitiation and read-through, followed by
the 5
UTR (nucleotides 1-336) of the HIV-1 vRNA (NL-4.3
clone) ( 4 ). First, we sought to determine if endogenous hn-
RNPK modulates the activity of the HIV-1 IRES. For this,
HEK293T cells were transfected with the dl HIV-1 IRES plas-
mid, a scrambled (sc) RNA (200 nM) or DscRNA (10 nM)
control, or a siRNAK (Figure 3 B–D) or DsiRNAK (Figure 3 E–
G). Wes t ern blot analysis conrmed that treating cells with
either the siRNAK or DsiRNAK reduced hnRNPK (Figure
3 B and E). As an aggressive knockdown of hnRNPK can in-
duce cell death ( 53 ), we decided to verify cell viability. The re-
sults showed that the level of reduction in hnRNPK induced
by either the siRNAK or DsiRNAK did not affect cell viabil-
ity ( Supplemental Figure S2 A). Luciferase activities were then
measured and expressed as relative luciferase activity (RLA),
with the RLuc and FLuc levels from cells transfected with the
negative controls, scRNA or DscRNA, set to 100% (Figure 3 C
and F). A signicant decrease ( ∼48%) of FLuc with a slight in-
crease of RLuc ( ∼13%) was observed in cells transfected with
the dl HIV-1 IRES plasmid and treated with the siRNAK (Fig-
ure 3 C). Similar observations were made when the DsiRNA
was used (Figure 3 E), with a slight increase in RLuc ( ∼11%)
activity and a signicant decrease in FLuc activity ( ∼44 %).
As RLuc activity did not decrease, these results suggest that
the reduction of FLuc activity when hnRNPK is targeted by
either the siRNAK or the DsiRNAK cannot be attributed to
the reduced stability of the dl HIV-1 IRES mRNA (Figure 3 C
and F). Analysis of the FLuc / RLuc ratio (relative translational
activity, RTA), conrmed the signicant reduction in HIV-1
IRES activity in cells treated with either siRNAK ( ∼52%) or
DsiRNAK ( ∼51%) (Figure 3 D and G).
To further validate the association between HIV-1 IRES ac-
tivity and hnRNPK levels, HEK293T cells were transfected
with the dl HIV-1IRES plasmid with the DsiRNAK (10 mM)
RNA and an irrelevant control DNA (200 ng) or with in-
creasing concentrations (50–200 ng) of a plasmid encoding
a hemagglutinin (HA
3
)-tagged hnRNPK, HA-hnRNPK. The
HA-hnRNPK mRNA lacks the hnRNPK mRNA 3
UTR and,
therefore, is not susceptible to DsiRNAK RNA. As expected,
treatment of cells with the DsiRNAK RNA reduced endoge-
nous hnRNPK levels (Figure 3 H, lane 2), also RLuc activity
slightly increased ( ∼10%), while FLuc activity decreased sig-
nicantly ( ∼46%) (Figure 3 I). In cells expressing the DsiR-
NAK RNA and HA-hnRNPK (Figure 3 H, lanes 3–5), FLuc
activity was restored (Figure 3 I). When data are presented as
RTA, DsiRNAK treatment led to reduced HIV-1 IRES activity
( ∼51%) in cells, but HIV-1 IRES activity was restored when
the hnRNPK expression was rescued (Figure 3 J). Thus, we
conclude that HA-hnRNPK promotes HIV-1 IRES activity in
HEK293T cells.
HnRNPK overexpression stimulates HIV-1 IRES
activity
Next, HEK293T cells were transfected with the dlHIV-1 IRES
plasmid, an irrelevant DNA (negative control), or different
concentrations (50, 100 or 200 ng) of the HA-hnRNPK plas-
mid. The overexpression of HA-hnRNPK was monitored by
western blot using an anti-hnRNPK or an anti-HA antibody,
using GAPDH as a loading control (Figure 4 A). The overex-
pression of HA-hnRNPK did not appreciably impair cell vi-
ability (Figure 4 B). Luciferase activities were measured, and
data were expressed as RLA, with the values of the luciferase
activities obtained from cells transfected with the control
DNA (–) set to 100% (Figure 4 C). A dose-dependent increase
in FLuc activity with increasing HA-hnRNPK (50–200 ng of
DNA) was observed (Figure 4 C). While there was little impact
on RLuc activity at 50 ng HA-hnRNPK DNA, with a signi-
cant increase of RLuc with 100 ng HA-hnRNPK DNA, the in-
crease in RLuc activity was highest when 200 ng HA-hnRNPK
was transfected in cells (Figure 4 C). Nonetheless, the increase
in RLuc activity was lower than that of FLuc, as evidenced
when the FLuc / RLuc ratio (RTA) is analyzed (Figure 4 D).
The increase in RLuc and Fluc could be due to the stabiliza-
tion of the HIV-1 IRES RNA. To evaluate this possibility, to-
tal RNA was extracted, and the relative amount of RLuc and
FLuc encoding RNA in the presence or the absence of HA-
hnRNPK, was independently determined. Results from these
analyses show that the presence of HA-hnRNPK did not in-
duce a signicant change in RNA either determined by RLuc
(39%) of FLuc (34%) RNA quantication (Figure 4 E). Thus,
changes to the stability of the dl HIV-1 IRES RNA cannot
explain the increase of FLuc over RLuc protein expression.
These results conrm that the overexpression of hnRNPK
stimulates HIV-1 IRES activity from the dl HIV IRES RNA.
As an additional control, we assessed the impact of HA-
hnRNPK on RLuc and FLuc expression from vector dl HIV-1
(1–104), which harbors the upstream sequence of the HIV-1
vRNA 5´UTR (nts 1–104) that is devoid of IRES activity ( 4 ).
Plasmid dl HIV-1 IRES and dl HIV-1 (1–104) only vary in the
segment of 5
UTR of the HIV-1 within the intercistronic space
( 4 ). The overexpression of hnRNPK signicantly increased the
expression of RLuc ( ∼89%) but did not correlate with an in-
crease in FLuc activity (Figure 4 F). These results conrm that
the expression of RLuc and FLuc are independent and that
upstream events leading to an increased RLuc expression do
not contribute to FLuc expression. Furthermore, these results
conrm that an active IRES is required to generate FLuc from
the dl HIV-1 IRES mRNA.
The overexpression of HnRNPK does not enhance
cryptic promoter activity from the dl HIV-1 IRES
DNA nor induces alternative splicing from the dl
HIV-1 IRES RNA in cells
The dl HIV-1 IRES reporter plasmid displays cryptic promoter
activity in HEK293T cells ( 21 ,22 ). Therefore, we sought
to determine if the overexpression of hnRNPK increased
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Nucleic Acids Research , 2024 9
AB
D
C
E
FG
H
IJ
Figure 3. Reduction of endogenous hnRNPK levels in cells negatively impacts HIV-1 IRES activity. ( A ) Schematic representation of dl HIV-1 IRES mRNA.
The capped and polyadenylated dual-luciferase (dl) bicistronic mRNA presents an upstream Renilla luciferase (RLuc) ORF and a downstream rey
luciferase (FLuc) ORF. A deleted 5
UTR of the encephalom y ocarditis virus ( EMCV) f ollo w ed b y the 5
UTR (nucleotides 1-336) of the HIV-1 vRNA, NL-4.3
clone, are placed between both cistrons. ( B–G ) HEK293T cells were cotransfected with the dl HIV-1 IRES (200 ng) mRNA encoding plasmid, a scRNA
(200 nM), or a siRNAK ( B-D ) targeting hnRNPK or with a DscRNA (10 nM) control or DsiRNAK (E–G). Reduction of endogenous hnRNPK was monitored
b y w estern blot using G APDH as a loading control ( B and E ) . RLuc and FLuc activities wer e measured at 24 hpt, and data are presented as RLA ( C and F )
or as RTA ( D and G ). RTA corresponds to the FLuc / RLuc ratio that is used as an index of IRES activity. The RLA and RTA values obtained in the absence
of HA-hnRNPK plasmid were set to 100%. ( H–J ) HEK293T cells were transfected with the dl HIV-1IRES plasmid with an irrelevant control DNA (200 ng)
and DsiRNAK (10 mM) alone or with increasing concentrations (50-200 ng) of a plasmid encoding for a HA-hnRNPK. The levels of endogenous and
o v ere xpressed hnRNPKs were monitored by western blot ( H ). RLuc and FLuc activities were measured at 24 hpt, and data are presented as RLA ( I ) or
as RTA ( J ). The RLA and RTA values obtained in cells transfected with the dl HIV-1 IRES plasmid and the DscRNAK RNA were set to 100%. Values
represent the mean ( ±SEM) from three independent experiments, with each conducted in duplicate. Statistical analysis was performed by performed
for ( C, D ) by Student’s t -test (* P < 0.05) and ( F, G ) and ordinary one-way ANO V A test (** P < 0.005, **** P < 0.0001; ns, not signicant)
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10 Nucleic Acids Research , 2024
A
CD
B
F
E
Figure 4. Ov ere xpression of hnRNPK promotes HIV-1 IRES activity. (A-D) HEK293T cells w ere cotransfected with the dl HIV-1 IRES (200 ng) and
different quantities (50-200 ng) of a plasmid encoding for an HA-hnRNPK protein. ( A ) The presence of the endogenous hnRNPK and overexpressed
HA-hnRNPK proteins was conrmed by western blot using GAPDH as a loading control. ( B ) Cell viability was determined by measuring the cellular
met abolic activit y using the MTS assa y using dimeth ylsulf o xide (DMSO, 10%) as a control f or cell death. Data are e xpressed relativ e to the viability of
the cells transfected with control DNA ( −) set to 100%. Values shown are the mean ( ±SEM) from three independent experiments, with each performed
in duplicate. Statistical analysis was performed by an ordinary one-way ANO V A test (*** P < 0.001). ( C, D ) . RLuc and FLuc activities were measured at
24 hpt, and data are presented as RLA ( C ) or as RTA ( D ). The RLA and RTA values obtained in the absence of HA-hnRNPK plasmid were set to 100%.
Values shown are the mean ( ±SEM) from four independent experiments, with each performed in duplicate. Statistical analysis was performed using
ANO V A, f ollo w ed b y Dunnet’s test (* P ≤0.05; ** P ≤0.0 1; *** P ≤0.00 1; ns = nonsignicant). ( E ) Cytoplasmic RNA w as e xtracted from cells
transfected with dlHIV-1 IRES and pCMV3-HA-hnRNPK or with the control plasmid, pSP64-polyA. The relative amount of RNA was determined by
R T- q P C R assa y. T he relativ e RNA abundance w as obtained b y setting the v alue obt ained from the cells transfected only with pSP64-polyA as 10 0% for
both RLuc and FLuc. Values represent the mean ( ±SEM) from four independent experiments, with each performed in duplicate. Statistical analyses
w ere perf ormed b y an unpaired tw o-tailed t -test (ns = nonsignicant). ( F ) HEK293T cells were cotransfected with the dl HIV-1 (1 –1 04) DNA and a
plasmid (200 ng) encoding for a HA-hnRNPK protein. RLuc and FLuc activities were measured 24 hpt, and data are presented as relative light units
(RLU). Values shown are the mean ( ±SEM) from three independent experiments, with each condition performed in duplicate. Statistical analysis was
performed using ANO V A, followed by Dunnet’s test (* P ≤0.05).
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Nucleic Acids Research , 2024 11
cryptic promoter activity within the dl HIV-1 IRES plas-
mid in HEK293T cells. For this, cells were transfected with
the dl HIV-1 IRES or the SV40 dl HIV-1 IRES plasmid
in the presence or absence of HA-hnRNPK. The expression
of HA-hnRNPK was monitored by western blotting of total
cell lysates using antibodies directed against the HA-tag and
GAPDH as a loading control (Figure 5 A). In the absence of the
SV40 promoter ( SV40; (Figure 5 A) and ( 21 ,22 )), RLuc and
FLuc activities from the reporters were signicantly ( P < 0.05)
diminished in cells (Figure 5 A). In agreement with earlier re-
ports ( 21 ,22 ), RLuc activity was detected in HEK293T, con-
rming both the leakiness of the experimental system and
the previously reported weak cryptic promoter activity of the
dl-plasmid in HEK293T cells ( 21 ,22 ). The presence of HA-
hnRNPK did not further modulate RLuc or FLuc expression
from the SV40 dl HIV-1 IRES plasmid, indicating that the
overexpression of HA-hnRNPK did not enhance cryptic pro-
moter activity from the dl HIV-1 IRES plasmid in HEK293T
cells (Figure 5 A).
HnRNPK is involved in HIV-1 pre-mRNA splicing ( 27 ). To
evaluate if the overexpression of HA-hnRNPK induced an al-
ternative splicing event leading to the synthesis of a FLuc en-
coding monocistronic mRNA, the Renilla ORF of the dl HIV-
1 IRES mRNA was targeted with a short interfering RNA, siR-
Luc (Figure 5 B, upper panel) ( 21 ,22 ). Alternatively, HEK293T
cells were cotransfected with the dl HIV-1 IRES and a con-
trol scRNA (Figure 5 B). The expression of HA-hnRNPK was
conrmed by western blot analyses using an anti-HA or anti-
hnRNPK antibody and GAPDH as a loading control (Figure
5 B, middle panel). In the presence of the siRLuc RNA, both
RLuc and FLuc activities were signicantly reduced, whether
HA-hnRNPK was overexpressed or not (Figure 5 B, lower
panel). When directly compared, the reduction of RLuc and
FLuc activities induced by the siRLuc RNA in the presence or
absence of overexpressed HA-hnRNPK protein was not sta-
tistically different (Figure 5 B, lower panel). This observation
conrms that the overexpression of HA-hnRNPK did not fa-
vor the generation of a FLuc-expressing monocistronic tran-
script from the dl HIV-1 IRES RNA.
ITAF activity of hnRNPK is modulated by
post-translational modication
In cells, the biological functions of hnRNPK are regulated
by post-translational modications (PTMs) ( 54–56 ). Thus, we
wondered if hnRNPK PTMs impacted the protein’s ability to
exert its ITAF function over the HIV-1 IRES. We selected and
evaluated the impact of phosphorylations on residues serine
(S) 216, S256, S284, S353 and tyrosine (Y) 458. We also in-
cluded methylations on residues arginine (R) 256 and R299.
PTM selection was based on reports associating the modi-
cations with hnRNPK intracellular distribution, RNA afn-
ity, and protein–protein interaction and their potential im-
pact on mRNA translation ( 41 , 42 , 54 , 57–61 ). In the case of
phosphorylation, S or Y residues were mutated to alanine (A)
(non-phosphorylated) or aspartic acid (D) (phosphomimetic).
For methylations, R residues were substituted by lysine (K).
HEK293T cells were transfected with the dl HIV-1 IRES and
the HA-hnRNPK or mutant HA-hnRNPK (mut-hnRNPK)
plasmids. The overexpression of the HA-hnRNPK and mut-
hnRNPK proteins, conrmed by western blotting, using an
anti-hnRNPK (total protein) or an anti-HA antibody (recom-
binant) and GAPDH as a loading control (Figure 6 A), did
not affect cell viability (Figure 6 B). Consistent with our pre-
vious results (Figures 4 C and 4 D), the overexpression of the
HA-hnRNPK (wt) signicantly increased HIV-1 IRES activ-
ity ( ∼75% increase) over the control ( −) (Figure 6 C). When
compared to HA-hnRNPK, none of the tested mutant pro-
teins abrogated HIV-1 IRES activity (Figure 6 C), however, the
mut-hnRNPK differentially promoted, or not, the activity of
the HIV-1 IRES. Stimulation of the HIV-1 IRES by the mu-
tants over the control ( −) was as follows: HA-mut-hnRNPK
S216A ( ∼91% increase) > S284A = S284 / S353A (78% in-
crease) > S284D = S353A = R256K ( ∼58% increase) (Fig-
ure 6 C). The HA-mut-hnRNPK S353D , S216D , S284 / 353D ,
Y458A, Y548D, R299K and R256 / 299K did not signicantly
stimulate HIV-1 IRES activity (Figure 6 C).
Next, we focused on the hnRNPK mutants S216D and
S284 / 353D, which lost the ability to stimulate HIV-1 IRES
activity. Earlier reports showed that the S284 / 353D mutant
is mainly cytoplasmic, while the S284 / 353A mutant is nu-
clear ( 41 ,62 ). Thus, the subcellular localization of S284 / 353D,
S284 / 353A, S216A and S216D mutants was evaluated by IF
in HEK293T cells. In agreement with previous reports ( 41 ,62 ),
S284 / 353D was identied in the cytoplasm, while S284 / 353A
remained mainly nuclear ( Supplemental Figure S3 A). IF re-
sults also show that the S216A mutant was mainly nu-
clear, while the S216D mutant was found in the cytoplasm
( Supplemental Figure S3 ). This nding was further conrmed
through a nuclear / cytoplasmic fractionation approach fol-
lowed by western blot analysis using PARP as a marker for
nuclear (N) extracts and GAPDH as a marker for cytoplas-
mic (C) extracts ( Supplemental Figure S3 B). Even though the
molecular mechanism remains unclear (discussed below), our
results support the notion that serine phosphorylation at S216
and S284 / 355 modulates hnRNPK’s ability to promote HIV-1
IRES activity . Interestingly , we nd that the nuclear localiza-
tion of hnRNPK (found with mutants S216A and S284 / 353A)
correlated with an increased HIV-1 IRES activity.
The phosphorylation of residue Y458, located within the
nucleic acid-binding site of the KH3 domain, is expected to re-
duce, but not abolish, hnRNPK interaction with target mRNA
( 42 ,60 ). Unexpectedly, our results showed that Y458A ex-
hibited effects similar to Y458D ( 42 ) on hnRNPK’s capac-
ity to stimulate HIV-1 IRES activity. To further understand
this apparent paradox, hnRNPK residue Y458 was mutated
to S, glutamic acid (E), arginine (R), phenylalanine (F) or
threonine (T). A previous report showed that hnRNPK mu-
tant Y458F binds RNA to the same extent as the wild-type
protein ( 42 ). HEK293T cells were transfected with the dl
HIV-1 IRES and HA-hnRNPK or Y458 mut-hnRNPK plas-
mids. The overexpression of the HA-hnRNPK and Y458 mut-
hnRNPK, as conrmed by western blot analyses, using an
anti-hnRNPK (total protein) or an anti-HA antibody (recom-
binant) and GAPDH as a loading control (Figure 6 D), did not
affect cell viability (Figure 6 E). As expected the HA-hnRNPK
overexpression stimulated ( ∼56% increase) HIV-1 IRES ac-
tivity, while Y458A, Y458D, Y458E, Y458S yielded modest
18%, 10%, 5% and 6% increases, respectively (Figure 6 F).
Y458R ( ∼11% decrease) induced a slight reduction in HIV-
1 IRES activity. Mutants Y458F and Y458T enhanced HIV-
1 IRES by 26% and 49%, respectively, but to a lower ex-
tent than wt-hnRNPK (Figure 6 F). The results are discussed
below.
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12 Nucleic Acids Research , 2024
AB
Figure 5. Ov ere xpression of hnRNPK does not enhance cryptic promoter activity of the dl HIV-1 DNA or induce alternativ e splicing of the dl HIV-1 IRES
RNA. ( A ) HEK 293T cells were transfected with either the dl HIV-1 IRES (150 ng) or a promoterless SV40-dl HIV-1 IRES (150 ng) vector in the presence
or the absence ( −) of the HA-hnRNPK (100 ng) plasmid. 24 hpt total protein extracts were prepared. A schematic representation of the dl HIV-1 IRES and
SV40-dl HIV-1 IRES plasmids is shown (upper panel). The expression of the HA-hnRNPK recombinant protein was determined by western blot, using
the GAPDH protein as a loading control (middle panel). RLuc and FLuc activities were measured, and results are expressed as RLA relative to the
activities obtained from the dl HIV-1 IRES vector when in the absence of the HA-hnRNPK, set to 10 0 % (lo w er panel). Values shown are the mean
( ±SEM) from three independent experiments, with each performed in duplicate. Statistical analysis was performed by an ordinary two-way ANO V A test
(**** P < 0.0 0 01; ns, nonsignicant). ( B ) The dl HIV-1 IRES (20 0 ng) was cotransfected with a control scRNA (50 nM) or with siRLuc (50 nM), in the
presence or the absence ( −), of the HA-hnRNPK (200 ng) plasmid. A schematic representation of the dl reporter targeted by the siRNA RLuc (siRLuc)
targeting the Renilla luciferase ORF is shown (upper panel). Tot a l protein extracts we re prepared 48 hpt. The expression of the HA-hnRNPK was
determined by we stern blot, using the GAPDH protein as a loading control (middle panel). RLuc and FLuc activities were measured and expressed
relative to the values obtained with scRNA, set to 100% (RLA) (lo w er panel). Values shown are the mean ( ±SEM) from three independent experiments,
with each performed in duplicate. Statistical analysis was performed by an ordinary one-way ANO V A test (* P < 0.05; ns, nonsignicant).
PRMT1-induced asymmetrical dimethylations
impact HIV-1 gene expression and the activity of
the HIV-1 IRES in HEK293T cells
Protein arginine methyltransferase 1 (PRMT1) is the only en-
zyme responsible for asymmetrically dimethylating hnRNPK
on mainly ve specic arginine residues (256, 258, 268,
296 and 299) ( 44 ). Since global PRMT1-induced arginine
methylations do not impact the translational activity of cel-
lular IRESs ( 63 ), nor do they affect various functions associ-
ated with hnRNPK, including RNA binding ( 44 ,55 ), we antic-
ipated that PRMT1-induced methylations of hnRNPK would
not alter the protein’s stimulatory effect over the HIV-1 IRES
activity. Contrary to our expectations, our results suggested
that PRMT1-induced asymmetrical dimethylations of argi-
nine residues (aDMAs) of hnRNPK are highly relevant for
HIV-1 IRES activity (Figure 6 C). If so, it was plausible to sus-
pect that aDMAs of hnRNPK could also impact HIV-1 gene
expression.
In our effort to establish if PRMT1 regulates HIV-
1 protein synthesis, the impact of N ,N
-(Sulfonyldi-4,1-
phenylene)bis(2-chloroacetamide) (TC-E 5003) ( 64 ), a selec-
tive PRMT1 inhibitor (IC
50
= 1.5 μM) on HIV-1 gene expres-
sion was evaluated. For this, HEK293T cells were transfected
with pNL4.3-RLuc (200 ng) and treated or not (vehicle alone)
with TC-E 5003 (0.125-2 μM). The treatment of HEK293T
cells with low TC-E 5003 concentrations between 0.125-2
μM did not impact cell viability ( Supplemental Figure S2 C),
but higher concentrations of TC-E 5003 (4-8 μM) signi-
cantly reduced cell viability ( Supplemental Figure S2 B). En-
dogenous aDMA levels of cellular proteins were monitored
by western blot analyses using a commercial mix of anti-
bodies that recognizes endogenous aDMA (Figure 7 A). The
treatment of cells with TC-E-5003 (0.125–2 μM) markedly
reduced endogenous aDMA signals at drug concentrations
of (0.5, 1 and 2 μM; Figure 7 A, lanes 4, 5 and 6) with-
out an evident impact on endogenous GAPDH control or
the abundance of endogenous hnRNPK (Figure 7 A). Wes t -
ern blot analysis using an anti-HA antibody revealed a sharp
reduction of Gag-RLuc-HA protein in cells treated with 1
and 2 μM TC-E-5003 (Figure 7 A, lanes 5 and 6). Consistent
with this observation RLuc activity was also signicantly re-
duced in cells treated with 1 μM ( ∼46% reduction) or 2 μM
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Nucleic Acids Research , 2024 13
A
BC
D
EF
Figure 6. hnRNPK post-translational modications impact on HIV-1 IRES activity. HA-hnRNPK mutants (mut) for the phosphorylation or methylation
sites were generated by site-directed mutagenesis of the HA-hnRNPK template plasmid (wt). HEK293T cells were cotransfected with the dl HIV-1 IRES
(200 ng) together with each HA-hnRNPK (200 ng) plasmid (indicated in the X-axis). To t a l protein extracts were prepared 24 hpt. ( A ) Weste rn blots wer e
performed to detect the expression level of total hnRNPK and HA-hnRNPK proteins using GAPDH as a loading control. ( B ) Cell viability was determined
by measuring the cellular metabolic activity using the MTS assay using dimethylsulfoxide (DMSO, 10%) as a control for cell death. Data are expressed
relative to the viabilit y of the cells transfected with control DNA ( −) set to 100%. Values shown are the mean ( ±SEM) from three independent
experiments, with each performed in duplicate. Statistical analysis was performed using ANO V A, followed by Dunnet’s test (*** P ≤0.001;
ns = nonsignicant). ( C ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities in the presence of the
HA-hnRNPK (wt), set to 100%. Values shown are the mean ( ±SEM) from ve independent experiments, with each performed in duplicate. Statistical
analy sis w as perf ormed using ANO V A, f ollo w ed b y Dunnet’s test (* P ≤0.05; ** P ≤0.0 1; *** P ≤0.00 1; ns, nonsignicant). (D–F) HEK293T cells were
cotransfected with dl HIV-1 -IRES plasmid (200 ng) and a plasmid expressing HA-hnRNPK(wt) or the Y458 mutants (200 ng). ( D ) Proteins were
monitored b y w estern blot analy sis using G APDH as a loading control. ( E ) Cell viability was determined as in (B); values are the mean ( ±SEM) from three
independent experiments, with each performed in duplicate. Statistical analysis was performed using ANO V A, followed by Dunnet’s test (*** P ≤0.001;
ns = nonsignicant). ( F ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities obtained from the dl HIV-1
IRES vector transfected with the control DNA, set to 100%. The values are presented as the average ( ±SEM) from three independent experiments, with
each performed in duplicate. Statistical analysis was performed using ANO V A, followed by Dunnet’s test (ns, nonsignicant; * P ≤0.05; ** P ≤0.01;
*** P ≤0.001; **** P ≤0.0001).
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14 Nucleic Acids Research , 2024
AB
E
D
C
FG
Figure 7. Inhibition of PRMT1 asymmetrical dimethylation impairs HIV-1 IRES activity. HEK 293T cells were cotransfected with the pNL-4.3-RLuc (200
ng) plasmid and 4 hpt treated, or not (only vehicle; ( −)), with TC-E 5003 (0.125, 0.25, 0.50, 1 or 2 μM), a specic inhibitor of the PRMT1 activity. To t al
protein extracts were prepared 24 h post-treatment. ( A ) The PRMT1 activity (aDMA), and levels of the HIV-1 Gag-RLuc-HA fusion protein and
endogenous hnRNPK in extracts from the untreated cell (lane 1) or from cells treated with at the different drug concentrations (0.125, 0.25, 0.50, 1 or 2
μM) wer e evaluated by western blot using GAPDH as a loading control. ( B ) RLuc activity was measured in proteins recovered from cells transfected
with the pNL-4.3-RLuc plasmid and treated with 1 or 2 μM of TC-E-50 03, and dat a are presented as RLA to the non-treated ( −) cells set to 100%. Values
shown are the mean ( ±SEM) from three independent experiments, with each performed in duplicate. Statistical analysis was performed using ANO V A,
f ollo w ed b y Dunnet’s test (** P ≤0.01; ns, nonsignicant). ( C–E ) HEK293T cells w ere transfected with the dl HIV-1 IRES (200 ng) plasmid, and 4 hpt
were treated, or not (only vehicle; ( −)), with TC-E 5003 (1, or 2 μM). To t a l protein extracts were prepared 24 h post-treatment. ( C ) The PRMT1 activity
(aDMA) and le v els of endogenous hnRNPK in extracts from the untreated cell (lane 1) or from cells treated with 1 or 2 μM of TC-E 5003 were evaluated
b y w estern blot using G APDH as a loading control. ( D, E ) RLuc and FLuc activities were measured, and data are presented as RLA ( D ) or RTA ( E ) relative
to the non-treated ( −) cells set to 100%. Values shown are the mean ( ±SEM) from six independent experiments, with each performed in duplicate.
Statistical analysis was performed using ANO V A, followed by Dunnet’s test (* P ≤0.05; ** P ≤0.01; *** P ≤0.001; ns, nonsignicant). ( F , G ) An
HA-hnRNPK mutant, 5RK, with the ve arginine residues (256, 258, 268, 296 and 299) targeted by PRMT1 substituted by lysine was generated by
site-directed mutagenesis of the HA-hnRNPK(wt) template plasmid (wt). HEK293T cells were cotransfected with the dl HIV-1 IRES (200 ng) together
with HA-hnRNPK(wt) (200 ng) or the 5RK (200 ng) plasmid. Tot a l protein extracts were prepared 24 hpt. ( F ) Wes ter n blots were performed to detect the
e xpression le v el of total hnRNPK and HA-hnRNPK proteins using G APDH as a loading control. ( G ) RLuc and FLuc activities w ere measured, and results
are presented as RTA, relative to extracts obtained from cells transfected with the dl HIV-1 IRES plasmid together with an irrele v ant DNA (pSP64-poly(A))
set to 100%. Values shown are the mean ( ±SEM) from ve independent experiments, with each performed in duplicate. Statistical analysis was
performed using ANO V A, followed by Dunnet’s test (* P ≤0.05; ** P ≤0.01; *** P ≤0.001).
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Nucleic Acids Research , 2024 15
( ∼68% reduction) of TC-E 5003 (Figure 7 B). These obser-
vations strongly suggest that PRMT1-induced aDMA plays a
role in HIV-1 gene expression.
Next, we directly assessed the effect of the selective PRMT1
inhibitor on HIV-1 IRES activity. For this, HEK293T cells
were transfected with the dl HIV-1 IRES plasmid (200 ng) and
treated, or not (vehicle alone), with TC-E 5003 (0.125–2 μM).
Global protein aDMA was followed by western blot analyses
(Figure 7 C). As shown earlier, the treatment of cells with TC-E
5003 (1 or 2 μM) reduced aDMA, with no apparent impact
on hnRNPK or GAPDH abundance (Figure 7 C). RLuc activity
in TC-E 5003 (1 or 2 μM) treated cells showed an increasing
trend ( ∼23% increase at 2 μM of the drug) (Figure 7 D). In
contrast, the FLuc activity was signicantly decreased ( ∼46%
reduction) at the highest concentrations of TC-E-5003 (2 μM)
(Figure 7 D). Results are also expressed as RTA to illustrate
better the signicantly lower activity ( ∼55% reduction) of
the HIV-1 IRES in cells treated with TC-E 5003 (2 μM) (Fig-
ure 7 E). As an additional control, cell extracts obtained from
HEK293T expressing RLuc and FLuc from the dl HIV-1 IRES
plasmid were directly mixed, or not, with TC-E 5003 (2 μM).
Results show that the PRMT1 inhibitor does not affect RLuc
or FLuc enzymatic activity ( Supplemental Figure S4 ).
The effect of TC-E 5003 was likely pleiotropic because
PRMT1 interacts with several proteins relevant to HIV-1 IRES
activity, such as eIF5A, Stau1, and hnRNPA1 ( 65 ). In addi-
tion, PRMT1 methylates hnRNPA1 ( 66 ). To directly address
whether PRMT1-induced aDMAs of hnRNPK impacted the
protein’ s IT AF function over the HIV-1 IRES, an HA-tagged
hnRNP K-5RK mutant (5RK) was constructed. In this mu-
tant, the ve arginine residues (256, 258, 268, 296,and 299)
targeted by PRMT1 were substituted by lysine. HEK293T
cells were transfected with the dl HIV-1 IRES DNA and
the plasmids expressing HA-hnRNPK or the HA-5RK mu-
tant. The overexpression of the 5RK, conrmed by west-
ern blot analyses (Figure 7 F), did not impact cell viability
( Supplemental Figure S2 C). The overexpressed 5RK remained
predominantly, but not exclusively, nuclear ( Supplemental
Figure S3 A). Luciferase activities were monitored, and re-
sults presented as RTA show that the overexpression of HA-
hnRNPK(wt) increased HIV-1 IRES activity, while the over-
expression of 5RK reduced HIV-1 IRES activity ( ∼35%)
(Figure 7 G).
Next, we decided to exclude the possibility that the ob-
served effect was due to the HA-tag or selected amino acid
substitutions used to mutate hnRNPK. Experiments were
therefore conducted in HEK293T cells using the previously
characterized GFP-hnRNPK and GFP-5RG mutant, in which
the arginine residues 256, 258, 268, 296 and 299 of hnRNPK
were substituted by glycine ( 44 ). In contrast to the GFP-
hnRNPK, the expression of the GFP-5RG mutant protein
( Supplemental Figure S5 A) non-signicantly reduced HIV-
1 IRES activity ( ∼25%) in HEK293T cells ( Supplemental
Figure S5 B), conrming that hnRNPK requires the PRMT1-
induced aDMAs to stimulate HIV-1 IRES activity.
We next sought to conrm that the HIV-1 IRES stim-
ulation by hnRNPK overexpression was also observed in
HeLa cells. For this, HeLa cells were transfected with dl
HIV-1 IRES or dl HIV-1 1-104 (lacking IRES activity) plas-
mid alone or in combination with plasmids encoding for
the HA-hnRNPK or the HA-5RK proteins. The expression
of HA-hnRNPK, the HA-5RK, FLuc, and RLuc, was con-
rmed by western blot using GAPDH as a loading con-
trol ( Supplemental Figure S6 A). As a negative control, non-
transfected cells (NT) extracts were also loaded in the west-
ern blot assay ( Supplemental Figure S6 A, lane 7). As expected,
RLuc was detected when cells were transfected with either the
dl HIV-1 1-104 ( Supplemental Figure S6 A, lanes 1–3) or the
dl HIV-1 IRES ( Supplemental Figure S6 A, lanes 4–6), while
FLuc was detected only when cells were transfected with the
dl HIV-1 IRES plasmid ( Supplemental Figure S6 A, lanes 4–6).
Co-transfection of HA-hnRNPK, but not HA-5RK, increased
the apparent amount of FLuc without impacting the amount
of RLuc ( Supplemental Figure S6 A, lanes 5–6). To further val-
idate our observations, HeLa cells were transfected with the dl
HIV-1 IRES plasmid with an irrelevant DNA ( −) or together
with plasmids encoding for HA-hnRNPK or the HA-5RK.
The expression of the recombinant proteins was conrmed by
IF ( Supplemental Figure S6 B). As anticipated, both the cap-
dependent RLuc, and the HIV-1 IRES-dependent FLuc pro-
teins could be readily detected by IF in HeLa cells transfected
with the dl HIV-1 IRES plasmid ( Supplemental Figure S6 B).
The mean uorescence intensity (MFI) values for RLuc and
FLuc obtained from the imaging data were used to calculate
the RTA as previously described ( 22 ). The RTA value obtained
in the absence of a recombinant protein ( −) was set to 100%.
Consistent with our previous observations, the overexpression
of HA-hnRNPK, but not HA-5RK, enhanced HIV-1 IRES ac-
tivity in HeLa cells ( Supplemental Figure S6 C). In conclusion,
PRMT1-induced aDMAs enable hnRNPK to stimulate HIV-1
IRES activity.
HnRNPK and the 5RK are equivalently associated
with the HIV-1 vRNA but interact differently with
known HIV-1 IRES ITAFs
To gain insight into the possible mechanism by which hn-
RNPK and 5RK exert their function over HIV-1 vRNA trans-
lation, HeLa cells were cotransfected with the HA-hnRNPK
or HA-5RK expression plasmids, and the pNL4.3 DNA. Vi-
ral replication was monitored by combined IF and uores-
cence in situ hybridization (IF / FISH ) for p24 and the vRNA
( Supplemental Figure S7 A) ( 67 ). The co-localization between
p24 ( Supplemental Figure S7 B) or the recombinant hnRNPK
proteins and the vRNA ( Supplemental Figure S7 C) was de-
termined by calculating the Mander’s coefcient per frame
and expressed as a percentage. Gag (p24) was equivalently co-
localized with the vRNA either in the presence or not of HA-
hnRNPK or the HA-5RK mutant ( Supplemental Figure S7 B).
In agreement with previous ndings showing that PRMT1-
induced arginine methylations of hnRNPK did not impact the
ability of the protein to bind its target RNA ( 44 ,55 ), both
HA-hnRNPK and HA-5RK equivalently co-localized with the
HIV-1 vRNA ( Supplemental Figure S7 C).
In cells, hnRNPK interacts with several known ITAFs
for the HIV-1 IRES, including hnRNPA1, DDX3, and HuR
( 33 ,34 ). Asymmetric arginine demethylation of hnRNPK by
PRMT-1 impacts the ability of hnRNPK to interact with
protein partners such as DDX-3, c-Src and p53 ( 44 ,68–70 ).
Therefore, we wondered whether hnRNPK and the 5RK mu-
tant equivalently interacted with known HIV-1 IRES ITAFs,
hnRNPA1, DDX3 and HuR (Figure 8 ). For this, HEK293T
cells were transfected with the dl HIV-1 IRES and the ex-
pression plasmid for HA-hnRNPK or HA-5RK, and 48 hpt,
co-immunoprecipitation (CoIP) assay were performed us-
ing protein G-agarose (PGA)-beads loaded with either an
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16 Nucleic Acids Research , 2024
A
B
C
Figure 8.
HnRNPK and 5RK differentially interact with other known ITAFs for the HIV-1 IRES. HEK293T cells were cotransfected with the dl HIV-1 IRES
DNA and the HA-hnRNPK or the HA-5RK encoding plasmids. 48 h later, cells were lysed, and immunoprecipitation (IP) assays were performed using
protein A / G agarose coated with IgG or the anti-HA antibody. The beads were washed extensively and incubated with loading buffer at 95ºC, and the
supernatant from whole-cell lysate (input; In) of each sample and IP fractions (anti-IgG or anti-HA) were used for western blotting. Western blotting was
performed using ( A ) anti-HA, anti-hnRNPA1, and anti-GAPDH antibodies, ( B ) anti-DDX3, anti-HA and anti-GAPDH antibodies, or ( C ) anti-HA, anti-HuR, and
anti-GAPDH antibodies. Horseradish peroxidase (HRP)-conjugated protein A / G was used to detect the primary antibodies .
anti-HA, or anti-IgG control antibody coupled with a west-
ern blot analysis. Proteins were detected in whole cell extracts
(input; In) from cells transfected with the respective express-
ing plasmid (Figure 8 ). As a loading control, GAPDH was
detected using an anti-GAPDH-specic antibody. A compari-
son of GAPDH in the input suggests that similar amounts of
protein were loaded (Figure 8 ). Capture and IP of the HA-
hnRNPK and HA-5RK were conrmed by western blotting
using an anti-HA antibody (Figure 8 ). HA-hnRNPK and HA-
5RK could be detected when the IP assay was performed using
PGA beads coated with the anti-HA, but not when beads were
covered with the anti-IgG control antibody (Figure 8 ). In the
presence of the dl HIV-1 IRES vector, endogenous hnRNPA1
(Figure 8 A), DDX3 (Figure 8 B), and HuR (Figure 8 C) co-
immunoprecipitated with HA-hnRNPK and HA-5RK. How-
ever, compared to HA-hnRNPK, DDX3 and HuR were poorly
enriched when HA-5RK was used (Figure 8 ).
HnRNPK is an ITAF for the HTLV-1 IRESs, but not for
the sHBZ IRES
Next, we questioned whether hnRNPK could also modulate
the activity of other retroviral IRESs. To address this issue,
we selected to assess the HTLV-1 IRES, present within the
5
UTR of the human T cell lymphotropic virus-type 1 (HTLV-
1) vRNA ( 37 ), and the sHBZ IRES, found within the 5
UTR
of the spliced version of the antisense HTLV-1 mRNA encod-
ing for the HTLV-1 basic leucine zipper protein (sHBZ) ( 38 ).
First, we sought to determine if hnRNPK interacts with the
5
leader of the HTLV-1 vRNA or the antisense mRNA encod-
ing sHBZ in vivo . HEK293T cells were transfected with dl
EMCV, dl HTLV-1 IRES or dl sHBZ IRES plasmids with
the HA-hnRNPK expressing plasmids. HEK293T cells were
transfected with dl EMCV and the pSP64 Poly(A) plasmid
as a control. Cells were treated with ultraviolet (UV) light
to covalently cross-linking (CL) proteins to RNA ( 46 ). Tot a l
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Nucleic Acids Research , 2024 17
proteins were recovered, and HA-hnRNPK was IP using PGA-
beads loaded with an anti-HA antibody. HA-hnRNP K was
detected in the input from transfected cells (Figure 9 A). As a
loading control, GAPDH was detected using an anti-GAPDH-
specic antibody. A comparison of GAPDH in the whole cell
extracts or input (In) suggests that similar amounts of protein
were loaded (Figure 9 A). Capture and IP of the recombinant
protein was conrmed by western blotting using an anti-HA
(Figure 9 A). HA-hnRNPK could be detected when the IP as-
say was performed using PGA beads coated with the anti-HA
antibody but not when anti-IgG coated PGA beads were used
(Figure 9 A). After the IP, proteins were removed by proteinase
K treatment, and FLuc RNA and GAPDH RNA were quan-
tied by real-time RT-qPCR ( 2 ,46 ). FLuc RNA and GAPDH
RNA were also quantied in the total input. The fold enrich-
ment (FE) of FLuc RNA and GAPDH RNA in the IP from
cells transfected with the dl EMCV in combination with the
pSP64 Poly(A) was set to 1 (unspecic pulldown / binding). FE
shows the enrichment of FLuc RNA or GAPDH RNA found
in the IP using the anti-HA antibody (specic IP) over the FLuc
RNA or GADPH RNA found in the IP using the anti-IgG con-
trol antibody (unspecic IP). The FLuc RNA or GAPDH RNA
from each IP was normalized to the FLuc RNA or GAPDH
RNA found in the input to account for any differences in the
RNA sample preparation ( 46 ). Results showed that dl HTLV-
1 IRES (FE ∼17) and dl sHBZ IRES (FE ∼5) RNAs were
signicantly enriched in the IPs using anti-HA antibody over
IPs with the anti-IgG control antibody, while GAPDH RNA
was not (Figure 9 B). Based on the UV-CLIP / RT-qPCR data,
we conclude that hnRNPK interacts with the 5
leader of the
HTLV-1 vRNA more efciently than with the shbz mRNA in
cells.
Next, we evaluated the impact of endogenous hnRNPK
knockdown on the activity of the HTLV-1 IRES and sHBZ
IRES by transfecting HEK293T with the dl HTLV-1 IRES (Fig-
ure 9 C) or dl sHBZ IRES (Figure 9 D) together with the DsiR-
NAK (10nM) or the DscRNA (10 nM) control RNA. Ta r g et-
ing the endogenous hnRNPK (Figure 9 C) reduced FLuc activ-
ity from the dl HTLV-1 IRES ( ∼48% decrease) without signif-
icantly impacting on RLuc activity (Figure 9 D). This observa-
tion is better supported when the data are presented as RTA
where the HTLV-1 IRES ( ∼50% reduction) activity decreased
in cells treated with the DscRNAK RNA (Figure 9 E). Inter-
estingly, the decrease of endogenous hnRNPK levels (Figure
9 F), did not impact the activity of the sHBZ IRES (Figure 9 G
and H).
To determine whether the overexpression of HA-hnRNPK,
which does not impact plasmids cryptic promoter activity or
induces alternative splicing of the dl-RNAs ( Supplemental
Figure S8 ), affected HTLV-1 and sHBZ IRES activity, the
dl HTLV-1 IRES (Figure 10 A–C) or dl sHBZ IRES (Figure
10 D–F) and the HA-hnRNPK (50, 100, 200 ng) expression
plasmids or an irrelevant DNA ( −) were co-transfected into
HEK293T cells. The expression of the HA-hnRNPK protein
was conrmed by western blot (Figure 10 A and D). Luciferase
activities were measured, and data were expressed as RLA
(Figure 10 B and E) or RTA (Figure 10 C and F). The overex-
pression of HA-hnRNPK did not alter RLuc activity in any of
the bicistronic mRNAs (Figure 10 B and E), but it stimulated
the activity from the HTLV-1 IRES ( ∼280% increase; 200 ng
DNA) (Figure 10 B). The impact of hnRNPK over the HTLV-1
IRES is better appreciated when the results are presented as
RTA (Figure 10 C). The overexpression of HA-hnRNPK had
no signicant effect over the sHBZ IRES (Figure 10 E and F).
These observations suggest that in HEK293T cells, hnRNPK
also acts as an ITAF for the HTLV-1 IRES without impacting
the activity of the sHBZ IRES.
Discussion
In this study, we identify hnRNPK, a member of the nuclear-
enriched poly (C)-binding protein (PCBP) family, as a bona
de ITAF that stimulates the activity of the HIV-1 (Figure 1 –
4 ) and HTLV-1 IRESs (Figures 9 and 10 ). HnRNPK deple-
tion reduces, while its overexpression stimulates activity from
the HIV-1 and HTLV-1 IRESs (Figures 3 , 9 and 10 ). Interest-
ingly, when compared to the 5
UTR of the HTLV-1 mRNA,
hnRNPK does not efciently bind the 5
UTR of the sHBZ
mRNA, nor does it act as an ITAF for the sHBZ IRES (Figures
9 and 10 ), suggesting that not all retroviral IRES require the
same subset of ITAFs for function. These ndings were not
unexpected as other ITAFs for retroviral IRESs are also virus-
specic. For example, the polypyrimidine-tract-binding pro-
tein (PTB) is needed for the activity of the IRESs present within
the Moloney murine leukemia virus and mouse mammary tu-
mor virus vRNAs ( 46 ,71 ), but it does not have a function in
translation initiation mediated by the HIV-1 IRES ( 25 ,46 ).
PTMs regulate the biological functions of hnRNPK ( 54–
56 ). In agreement, results from a basic screening approach
showed that the phosphorylation in S216, S284, S353 and
PRMT-1 induced asymmetrical dimethylation of hnRNPK im-
pacted the ability of the protein to stimulate HIV-1 IRES activ-
ity. As previously reported ( 41 ,62 ), the S284 / S353D hnRNPK
mutant accumulated in the cell cytoplasm ( Supplemental
Figure S3 ). Strikingly, when S284 / S353D hnRNPK was over-
expressed the stimulation of HIV-1 IRES activity was reduced
(Figure 6 ). In contrast, the S284 / S353A hnRNPK mutant,
which is mainly nuclear ( Supplemental Figure S3 and ( 62 )),
enhanced HIV-1 IRES activity (Figure 6 ). In cells, mitogen-
activated protein kinase / extracellular-signal-regulated kinase
(MAPK / ERK) phosphorylates hnRNPK at S284 and S353
( 41 ). Interestingly, hnRNPK translation-regulatory activity
dependent on phosphorylation of hnRNPK on S284 / 353
by MAPK / ERK is not exclusively observed for the HIV-1
IRES (Figure 6 ), as IRES-mediated translation initiation of
the c-myc mRNA is also increased when the wild-type and
S284 / 353A hnRNPK are overexpressed ( 30 ,72 ). In contrast,
when phosphorylated at S284 / 353, hnRNPK inhibits trans-
lation of the Lox mRNA ( 41 ). HnRNPK regulates gene ex-
pression by integrating the cross-talk between kinases and
hnRNPK-interacting partners ( 56 ). Therefore, it is not sur-
prising that PTMs, such as phosphorylation at S284 / 353 in
hnRNPK, differentially impact target mRNAs. This would
most likely occur through different mechanisms, presenting
hnRNPK as a central node for a complex translational control
network in normal physiology and during virus replication.
Regarding the amino acid in position 458 (Figure 6 ), a pre-
vious report showed that the substitution of amino acid 458
did not alter the structure of hnRNPK ( 42 ). This substitu-
tion, however, modied the chemical environment of the C-
terminal end of the protein, likely changing long-range inter-
actions of the tyrosine side chains with the core KH domain,
impacting the protein’s ability to interact with its target RNA
( 42 ). Although highly speculative, it is plausible that threo-
nine could partially restore the interaction of site 458 with the
core KH domain required to enable hnRNPK to function as an
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18 Nucleic Acids Research , 2024
AC
DG
F
EH
B
Figure 9. Reduction of endogenous hnRNPK negatively impacts HT LV-1 IRES without affecting sHBZ IRES activity. HEK 293T cells were transfected
with the dl EMCV, dl HTLV-1 IRES, or dl sHBZ IRES plasmids, proteins and RNAs were ultraviolet (UV) cross-linked (254 nm-irradiated with 400
mJ / cm
2
). Endogenous hnRNPK was immunoprecipitated (IP) from cell extracts using protein A / G-agarose (PGA)-beads loaded with an anti-hnRNPK
antibody or using PGA-beads loaded with an anti-IgG antibody as a negative control. ( A ) Th e hnRNPK protein present in the input extracts and the IPs
w ere e v aluated b y w estern blotting using an anti-HA antibody. As a loading control, G APDH w as detected using an anti-G APDH-specic antibody. T he
protein A / G HRP conjugated was used as the secondary antibody. ( B ) The quantity of FLuc encoding RNA (FLuc) or GAPDH encoding RNA (GAPDH)
co v alently bound to hnRNPK was determined by an RT-qPCR assay as described in ( 46 ). RNA fold enrichment, as dened in ( 46 ), obtained in the IP from
cells transfected with the dl EMCV DNA, was set to 1. Values shown are the mean ( ±SEM) from two independent experiments, with each RT-qPCR
assa y perf ormed in duplicate. Statistical analy ses w ere perf ormed using the ANO V A Kruskal-Wallis test ( P < 0.05). ( C-H ) HEK293T cells were
cotransfected with ( C–E ) the dl HT LV-1 IRES (200 ng), or ( F–H ) the dl sHBZ IRES (200 ng) plasmids, and a DscRNA (10 nM) control or DsiRNAK RNAs.
Reduction of endogenous hnRNPK was monitored by western blot using GA PDH as a loading control ( C and F ). RLuc and FLuc activities were measured
at 24 hpt, and data are presented as RLA ( D and G ) or as RTA ( E and H ). The RLA and RTA values obtained in the absence of when using the DscRNA
were set to 100%. Values represent the mean ( ±SEM) from three independent experiments, with each conducted in duplicate. Statistical analysis was
perf ormed b y a t-student test (ns, nonsignicant; * P < 0.05).
ITAF for the HIV-1 IRES activity. Based on our results (Figure
6 ), the impact of Y458 phosphorylation on HIV-1 IRES activ-
ity remains inconclusive as the chosen experimental approach
could not adequately assess it. Further experiments will, there-
fore, be necessary to fully understand the molecular mecha-
nism associated with the impact of residue 458 on the ability
of hnRNPK to stimulate HIV-1 IRES activity.
We included R256K, R299K and R256 / 299K mutants
when designing our experiments, because residue R256 par-
ticipates in hnRNPK interaction with the ribosomal protein
19 (RPS19) ( 68 ), and R299 is the dominant site of PRMT1
methylation ( 73 ). Our results suggest that PRMT-1 does not
impact HIV-1 IRES-mediated translation initiation by interfer-
ing with hnRNPK-40S ribosomal interaction (through RPS19)
as mutant R256K stimulated HIV-1 IRES activity (Figure 6 C).
What was unexpected was that R299K and R256 / 299K mu-
tants were unable to stimulate HIV-1 IRES activity (Figure
6 C), suggesting that hnRNPK PRMT1-induced aDMAs are
required to stimulate the activity of the HIV-1 IRES. This
observation was conrmed by pharmacological inhibition of
PRMT1 activity and using the 5RK mutant (Figure 7 and
Supplemental Figures S5 and S6 ). This nding was unexpected
since an earlier report showed that the pharmacological inhi-
bition of PRMT1 had no observable impact on the transla-
tional activity of the IRESs found in mRNAs encoding Cyclin
D1 (CCD1), cellular Myelocytomatosis (c-MYC), hypoxia-
inducible factor-1 α(HIF1 α), estrogen receptor α(ESR1), and
cyclin-dependent kinase inhibitors 1B (CDKN1B) ( 63 ). The
mechanism by which PRMT1 methylated hnRNPK affects
HIV-1 IRES activity remains unclear . However , in agreement
with reports indicating that PRMT1-induced aDMAs of hn-
RNPK do not inuence the protein’s ability to bind RNA
( 44 ,55 ), we observed that in HIV-1 replicating cells and over-
expressing the wt-hnRNPK or the 5RK mutant, both re-
combinant proteins co-localize with the vRNA equivalently
( Supplemental Figure S7 ). A possible clue to understanding
our results emerges from reports indicating that PRMT1-
induced aDMAs of hnRNPK affect the ability of the protein to
interact with some of its molecular partner’s regulating in this
way its biological function ( 44 ,68 ). In agreement with these re-
ports, we nd that in the presence of the dl HIV-1 IRES RNA,
the wt-hnRNPK and 5RK mutant differentially interact with
DDX3 and HuR, two known HIV-1 IRES ITAFs (Figure 8 ). In-
terestingly, and entirely consistent with an earlier report ( 68 ),
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Nucleic Acids Research , 2024 19
F
E
D
A
B
C
Figure 10.
Ov ere xpression of hnRNPK promotes the activity of the HTLV- 1 IRES but not from the sHBZ IRES. HEK293T cells w ere cotransfected with
( A–C ) the dl HTLV- 1 IRES (200 ng), or ( D–F ) or the dl sHBZ IRES (200 ng) plasmids, and the HA-hnRNPK encoding plasmid. The presence of the
o v ere xpressed HA-hnRNPK protein was conrmed by western blot using GAPDH as a loading control ( A and D ). RLuc and FLuc activities were
measured at 24 hpt, and data are presented as RLA ( B and E ) or RTA ( C and F ). The RLA and R TA v alues obtained in the absence of HA-hnRNPK plasmid
were set to 100%. Values represent the mean ( ±SEM) from three independent experiments, each conducted in duplicate. Statistical analysis was
perf ormed b y ANO V A (* P < 0.05).
showing that both DDX3 and HuR are enriched when IPs are
conducted using a methylated version of hnRNPK, we nd
when using the HA-5RK mutant DDX3 and HuR are less en-
riched (Figure 8 ). A potential role of the hnRNPK-DDX3 in-
teraction in translational control has not been reported. How-
ever, the hnRNPK-HuR complex, evidenced only in prolifer-
ating cells ( 33 ), regulates translation of the p21 mRNA ( 74 ).
Whether the decrease in 5RK interaction with DDX3 or HuR
observed by co-immunoprecipitation is solely responsible for
the reduced ability of the hnRNPK mutant to stimulate HIV-1
IRES remains inconclusive. Nonetheless, our results do high-
light the relevance of the ribonucleoprotein complex that as-
sembles on the HIV-1 vRNA to enable ne-tuning of IRES
activity.
The results obtained with the hnRNPK mutants suggest
a model that links nuclear events with the rate of cap-
independent translation of the HIV-1 vRNA. Noteworthy is
that hnRNPA1 associates with the vRNA in the nucleus and
is exported with the vRNA as part of an RNP to modu-
late HIV-1 IRES-mediated translation initiation ( 11 ). Thus,
it is tempting to speculate that aDMAs of hnRNPK medi-
ated by PRMT1 enhance specic nuclear protein–RNA and
protein-protein interactions, favoring the assembly of an ef-
cient translational RNP-complex required later to promote
HIV-1 IRES activity ( 1 ). In support of this possibility is the
model proposed for cellular IRESs that suggests the nuclear as-
sembly and transit to the cytoplasm of translation-competent
RNP complexes ( 2 , 30 , 75 , 76 ). An alternative possibility that
cannot be discarded is that the amount of endogenous hn-
RNPK found in the cytoplasm is sufcient to ensure HIV-1
IRES-mediated translation initiation. This last model is sup-
ported by the observation that HIV-1 gene expression does not
induce a redistribution of hnRNPK to the cytoplasm (Figure
2,Supplemental Figure S1 ). Adding further complexity to its
role in HIV-1 IRES-mediated translation initiation are several
reports that show that endogenous hnRNPK actively mod-
ulates the translation of cellular and viral mRNAs by us-
ing different molecular mechanisms. For some mRNAs, hn-
RNPK binds to their 3
UTR, impeding 80S ribosome assem-
bly. For others, hnRNPK binds to the 5
UTR, altering RNA
structure or affecting the recruitment of other proteins in-
volved in translation initiation ( 30 , 31 , 43 , 72 , 77–81 ). So, the
precise molecular mechanism hnRNPK uses to promote HIV-
1 IRES activity is difcult to anticipate without further work.
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20 Nucleic Acids Research , 2024
However, we suspect that the action of hnRNPK on HIV-1
IRES activity is linked with its ability to interact with other
partner proteins (Figure 8 ). Further experiments will be re-
quired to determine how hnRNPK interacting partners impact
HIV-1 IRES activity.
In cells, hnRNPK is a component of several dynamic com-
plexes whose composition changes in response to their loca-
tion and the cellular environment ( 33 ). Biological functions
ascribed to hnRNPK appear to depend on specic complexes
in which hnRNPK is found. For example, in the context of
HIV-1, as part of the Nef-associated kinase complex, hnRNPK
increases vRNA transcription ( 26 ), and when hnRNPK inter-
acts with Rev, it leads to the enhancement of Rev-mediated
RRE-dependent gene expression ( 28 ,29 ). HnRNPK has more
than 100 identied protein partners, some known to play a
role in translational control ( 33 ). In this regard, our ndings
open new avenues of research as several other proteins, known
to interact with hnRNPK to modulate translation initiation
of its substrate mRNAs ( 68 ), may also be relevant for HIV-1
IRES activity.
In conclusion, we provide evidence that hnRNPK acts as a
genuine ITAF for the HIV-1 and HTLV-1 IRESs, promoting
their activity and identifying a novel and additional function
of this multifunctional RBP in retroviral gene expression.
Data availability
All data are available upon request.
Supplementary data
Supplementary Data are available at NAR Online.
A c kno wledg ements
We thank Antje Ostareck-Lederer ( University Hospital
RWT H Aachen, Aachen, Germany), Ricardo Soto-Rifo (Uni-
versidad de Chile, Santiago, Chile), William Rigby (Dart-
mouth Medical School, NH, USA), and the NIH AIDS Refer-
ence and Reagent Program for sharing reagents. YF, Programa
de Doctorado en Microbiología, Universidad de Santiago, VO
and DM, Programa de Doctorado en Ciencias Médicas, Es-
cuela de Medicina, Facultad de Medicina, Ponticia Universi-
dad Católica de Chile, and BLU, Programa de Doctorado en
Ciencias Biológicas mención Genética Molecular y Microbi-
ología, Facultad de Ciencias Biológicas, Ponticia Universidad
Católica de Chile, conducted this work as part of their Ph.D.
Thesis. The funders had no role in the design of the study,
data collection, data analysis, the decision to publish, or the
preparation of the manuscript.
Author contributions : YF, VO, and HR performed experi-
ments showing that hnRNPK acts as an ITAF for HIV-1 IRES
in HEK293T cells. YF constructed hnRNPK mutants used in
the study and evaluated the role of phosphorylation’s of hn-
RNPK on its function as an ITAF for the HIV-1 IRES. VO, BLU
and HR performed experiments showing that hnRNPK acts as
an ITAF for HTLV-1 IRES. BLU conducted the CLIP assays for
the HTLV-1 and sHBZ IRESs and evaluated the impact of hn-
RNPK on the sHBZ IRES. ALA, GGC, and MN performed all
experiments involving HIV-1 gene expression in HeLa cells.
YF, ALA, and DM assessed the impact of PRMT-1-induced
asymmetrical dimethylations of arginines on hnRNPK func-
tion as an ITAF for the HIV-1 IRES. DM and BRA conducted
the CoIP assays. MLL conceived the study. MLL and AJM
supervised and secured funding for the study. MLL drafted
the manuscript, and all authors revised and approved the -
nal manuscript.
Funding
Agencia Nacional de Investigación y Desarrollo (ANID),
Gobierno de Chile [FONDECYT 1210736 to MLL]; Ini-
ciativa Cientica Milenio (ICM), Instituto Milenio de In-
munología e Inmunoterapia [ICM ANID ICN2021_045, IMII
to MLL]; Canadian Institutes of Health Research (CIHR)
[FRN-162447 to AJM]; YF (fellowship # 21191169), VO (fel-
lowship # 21150955), DM (fellowship # 21200983) and GGC
(fellowship # 72210500) were supported by ANID-Doctoral
Fellowships; BLU was supported by a Doctoral Fellowship
from the Vicerrectoria de Investigación (VRI), Ponticia Uni-
versidad Cátolica de Chile; BRA was supported by an IMII
(sub-área virus de RNA y vacunas) Post-Doctoral fellowship.
Funding for open access charge: ICM ANID ICN2021_045.
Conict of interest statement
None declared.
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Received: April 25, 2023. Revised: December 7, 2023. Editorial Decision: December 10, 2023. Accepted: December 15, 2023
©The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
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