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Single cycle infectivity of primary Envs derived from HIV-infected patients. Different Envs were used to generate pseudotyped virus particles that were used to determine relative infectivity in the TZM-bl cell line. A, bars represent pooled data (mean S.E. (error bars)) of duplicate or triplicate observations from three independent experiments. B, classification of infectivity of each primary Env based on high, medium, or low apoptosis inducers (*, p 0.05). C, classification of infectivity of each primary Env based on the virus subtype. D, graph depicting variation in apoptosis and infectivity of selected Envs.
Source publication
The Envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4+ T cell loss. However, most of the studies testing this phenomenon have been conducted with lab adapted HIV-1 isolates. This raises...
Contexts in source publication
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... and apoptosis induction are dependent on Env function, we next asked whether there were differences in the virion infec- tivity of the Envs studied. We used a pseudotyped virus particle approach to determine the single round infectivity of the Envs tested. Here again we found that there was a huge variation in the infectivity of the Envs tested (Fig. 4A). We found that high apoptosis-inducing Envs were more infectious compared with low apoptosis (p 0.05) (Fig. 4B), although subtype-based clas- sification did not show significant differences between the Envs (Fig. 4C). This is not surprising because both virion infectivity and bystander apoptosis are dependent on the Env fusion activ- ...
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... infec- tivity of the Envs studied. We used a pseudotyped virus particle approach to determine the single round infectivity of the Envs tested. Here again we found that there was a huge variation in the infectivity of the Envs tested (Fig. 4A). We found that high apoptosis-inducing Envs were more infectious compared with low apoptosis (p 0.05) (Fig. 4B), although subtype-based clas- sification did not show significant differences between the Envs (Fig. 4C). This is not surprising because both virion infectivity and bystander apoptosis are dependent on the Env fusion activ- ity. Although as a group, virus infectivity correlated with bystander apoptosis, this was not necessarily true ...
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... round infectivity of the Envs tested. Here again we found that there was a huge variation in the infectivity of the Envs tested (Fig. 4A). We found that high apoptosis-inducing Envs were more infectious compared with low apoptosis (p 0.05) (Fig. 4B), although subtype-based clas- sification did not show significant differences between the Envs (Fig. 4C). This is not surprising because both virion infectivity and bystander apoptosis are dependent on the Env fusion activ- ity. Although as a group, virus infectivity correlated with bystander apoptosis, this was not necessarily true for each Env. For instance, we found a number of Envs that showed relatively low apoptosis but very high ...
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... surprising because both virion infectivity and bystander apoptosis are dependent on the Env fusion activ- ity. Although as a group, virus infectivity correlated with bystander apoptosis, this was not necessarily true for each Env. For instance, we found a number of Envs that showed relatively low apoptosis but very high infectivity and vice versa (Fig. 4D). Hence, the spectrum of Envs studied here also includes a num- ber of Envs that may be highly infectious without causing sig- nificant apoptosis. This is consistent with several mutagenesis studies whereby point mutations in HIV Env significantly alter bystander apoptosis while having a limited effect on virion infectivity ...
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... further ranking can be seen by color transition from red to white to blue. Bank entry 2B4C) of GP120 (gray rib- bon) binding to CD4 (yellow ribbon) rendered using Yasara (55). Arg-476 (red residue) from gp120 is in close proximity with Gln-25 and Ser-23 (pink residues) from CD4. Asn-425 (blue residue) from GP120 is also within 5 Å of Phe-43 and Arg-59 (light blue residues) from CD4. ...
Citations
... Although these individuals are susceptible to HIV infection, they show delayed progression to AIDS possibly via lower CCR5 expression [18]. Our lab has extensively studied the role of CCR5 expression levels in HIV pathogenesis [19][20][21][22][23][24][25] and believe that a reduction in CCR5 levels in mono allelic KO may reduce CCR5 levels and provide a CCR5delta32 heterozygous like phenotype in patients resulting in reduced HIV pathogenesis. However, the goal of CCR5 KO should be bi allelic KO in order to achieve HIV resistance/cure as seen in the Berlin patient. ...
Background
Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone.
Methods
A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein.
Results
Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir.
Conclusions
Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.
... Previously, we and others have studied HIV Env mediated bystander apoptosis in coculture models whereby HIV Env expressing cells (effectors) induce apoptosis in CD4+ T cells (target) [18,[21][22][23][24]. Studies from these experiments have shown that the differential in vitro AIP of both HIV Env mutants and primary Env variants correlates with CD4 loss in the humanized mice model as well as in HIV patients [15][16][17]25]. To determine whether KB9 and 89.6 Envs have differential AIP we utilized the same coculture model system. ...
... Similarly, the fusogenic activity of SHIV Envs has been associated with CD4 decline in Macaque model of HIV infection [9]. Previous studies by our lab have shown that the apoptosis inducing potential or AIP of primary Envs derived from patients varies considerably [25]. More recently, we have found that this AIP of Envs correlates with CD4 decline with Envs with higher AIP associated with lower CD4 counts in patients [16]. ...
SHIV variants KB9 and 89.6 show differential pathogenesis in primate models with KB9 causing rapid CD4 decline while 89.6 failing to induce disease. We attempted to determine whether the differential pathogenicity of KB9 versus 89.6 was a result of differential bystander apoptosis inducing potential (AIP) of the Env glycoproteins from these viruses. We find that the KB9 Env was highly potent at inducing bystander apoptosis in CD4+ target cells compared to 89.6 Env. Cell death induction by KB9 showed classical signs of apoptosis including mitochondrial depolarization, caspase activation and PARP cleavage. Inhibiting Env mediated fusion by T20 peptide inhibited KB9 mediated bystander apoptosis. KB9 and 89.6 differed in terms of co-receptor usage with 89.6 preferring CXCR4 while KB9 using both CXCR4 and CCR5 with equal efficiency. Our study suggests that higher bystander AIP of KB9 Env compared to 89.6 may be the basis for the differential pathogenesis of these viruses.
... Several authors have described that Env showing similar fusogenicity may differ in other relevant functions such as cell death induction 16,32 . Therefore, we in-depth characterized different Env functions in our clones, first focusing on the binding of gp120 to CD4. ...
... Indeed, cell-to-cell HIV-1 transmission is able to activate different cell death mechanisms, classically associated with apoptosis, and more recently, with autophagy and pyroptosis 25,33,34 . All of them resulting in the rapid destruction of target cells before being productively infected (bystander cell death) 27,32 . In particular, Env triggers autophagy after cellular contacts between Env expressing and target cells 25 . ...
... independent of the HIV-1 + group (VNP or RP), it strongly correlated with the fusogenic capacity of Env, suggesting that autophagy induction promoted by primary isolates follows the same mechanisms described for laboratory adapted Env in T cells 24 . The bystander effect induced by Env has been associated to specific genetic signatures in residues 425 and 476 of gp120 32 and in residues in the HR1 sequence of gp41 16 . Consistent with the similar cytopathicity of Envs isolated from VNPs and RPs, no sequence differences in those residues were observed between both groups. ...
In untreated HIV-1-infected individuals, viremia is positively associated with disease progression. However, some viremic non progressors (VNPs) individuals show paradoxical high CD4+ T cell counts. HIV-1 envelope glycoprotein complex (Env) is a major cytopathic determinant in viral replication; therefore, we have deeply characterized Env function in this rare clinical phenotype. Full-length Env clones isolated from individuals with Viral Load (VL) > 10,000 copies/mL classified as VNPs (n = 15) or rapid progressors (RPs, n = 17) were geno- and phenotypically analyzed by determining diversity, expression, CD4 binding/signaling, fusogenicity, infectivity and autophagy induction. Selected Env clones from VNPs and RPs (n = 32) showed similar expression, fusion and infection abilities. Env clones from both groups showed similar affinity for CD4 during cell-to-cell transmission and consistently induced similar levels of CD4 signaling, measured by α-tubulin acetylation. Moreover, we demonstrate for the first time that primary Env clones from VNP and RP induce autophagy in uninfected cells and that this feature correlated with fusogenic capacity but was unrelated to disease progression. In conclusion, our data suggest that Env clones from VNP individuals are fully functional. Therefore, the paradoxical CD4+ T cell count stability coexisting with high levels of viral replication is unrelated to Env function.
... We and others have shown that CD4 apoptosis correlates with CD4 decline in HIV patients 24,[33][34][35] . Also, binding of HIV Env to CCR5 is required for bystander apoptosis induction 29,36 . We have also shown that levels of CCR5 on cell surface determines bystander apoptosis of cells via HIV Env, with higher CCR5 expression associated with increased bystander apoptosis in vitro 27,37 . ...
CCR5 is the major co-receptor for HIV and polymorphisms in the CCR5 gene as well as promoter region that alter cell surface expression have been associated with disease progression. We determined the relationship between CCR5 promoter polymorphisms and CD4 decline and other immunopathological features like immune activation and CD4+ T cell apoptosis in HIV patients. CCR5 promoter haplotype HHC was significantly associated with higher CD4 counts in patients. The relative promoter activity (RPA) of each haplotype was determined in vitro and combined promoter activity based on both alleles (CRPA) was assigned to each patients. Interestingly, CCR5 CRPA correlated inversely with CD4 counts and CD4:CD8 ratio specifically in viremic patients. In normal individuals, the CRPA correlated with the number of CCR5+ CD4+ T cells in the peripheral blood suggesting an effect on CCR5 expression. In a subset of high viremic patients harboring R5 tropic HIV, there was a strong correlation between CCR5 CRPA and both CD4 counts and CD4 T cell apoptosis. Our study demonstrates that, CCR5 promoter polymorphisms correlate with CD4 T cell loss possibly by regulating CD4 T cell apoptosis in HIV patients. Furthermore, assigning CRPAs to each patient is a new method of translating genotype to phenotype.
... First, a protective effect of these antiviral drugs in latently HIV-infected cells mediated by prevention of cell death of HIV-infected cells as described in the results, but there may be a second mechanism of cell protection of uninfected cells, by preventing binding of the virus to CD4 and CCR5. A similar mechanism of cell death protection mediated by CCR5 has been described in several cell types [73][74][75][76] . This point requires further exploration because this indicates that some ART may have negative effects on the survival of HIV reservoirs. ...
While HIV kills most of the cells it infects, a small number of infected cells survive and become latent viral reservoirs, posing a significant barrier to HIV eradication. However, the mechanism by which immune cells resist HIV-induced apoptosis is still incompletely understood. Here, we demonstrate that while acute HIV infection of human microglia/macrophages results in massive apoptosis, a small population of HIV-infected cells survive infection, silence viral replication, and can reactivate viral production upon specific treatments. We also found that HIV fusion inhibitors intended for use as antiretroviral therapies extended the survival of HIV-infected macrophages. Analysis of the pro- and anti-apoptotic pathways indicated no significant changes in Bcl-2, Mcl-1, Bak, Bax or caspase activation, suggesting that HIV blocks a very early step of apoptosis. Interestingly, Bim, a highly pro-apoptotic negative regulator of Bcl-2, was upregulated and recruited into the mitochondria in latently HIV-infected macrophages both in vitro and in vivo. Together, these results demonstrate that macrophages/microglia act as HIV reservoirs and utilize a novel mechanism to prevent HIV-induced apoptosis. Furthermore, they also suggest that Bim recruitment to mitochondria could be used as a biomarker of viral reservoirs in vivo.
... Increased affinity of gp120 for CD4 receptor and/or coreceptor can influence both Env mediated fusion as well as apoptosis [28, [57][58][59]. We have also found a negative correlation between potential N-glycosylation sites (PNGS) and bystander apoptosis inducing phenotype among primary patient derived Envs [60]. Thus, the phenotypic characteristic of Env glycoprotein is complex and a consequence of genotypic characteristics of both gp120 and gp41 subunits. ...
... We have found that in vitro adaptation of HIV to low levels of CCR5 results in evolution of virus to higher CCR5 affinity and increased bystander apoptosis in cell expressing low CCR5 levels [66]. An in vitro analysis of primary CCR5 tropic Envs for bystander apoptosis induction also shows that patient derived Envs vary considerably with respect to apoptosis phenotype and several genetic signatures correlated with apoptosis, including low levels of PNGS [60]. Recently, we have found that the apoptosis inducing potential (AIP) of primary Envs from patients correlates with CD4 decline and CD4:CD8 ratio in patients [10]. ...
... Recently, Env CCR5 interactions were also found to be important for bystander apoptosis induction via a dual tropic HIV isolate, with the process being reduced by CCR5 inhibitors or mutations in the Env glycoprotein that abrogate CCR5 interaction [29]. Thus, the process of bystander apoptosis in the larger context of HIV pathogenesis seems to rely on two key aspects, the Env fusogencity and host CCR5 expression levels [29,60]. Patients harboring highly fusogenic Envs would likely be efficient in using low levels of CCR5 while patients with less fusogenic Envs would require high CCR5 levels for bystander apoptosis induction. ...
Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis.
... We reasoned that this might have been caused by premature apoptosis of the packaging cells due to high levels of gp140. 29 We inserted the gp140 expression cassette into an E1-deleted viral molecular clone that contained the E3 domain. The rescued vector and target genome was initially changeable, however on subsequent passages remained stable. ...
Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert Env proteins of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing envelope (Env) protein of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable gp140- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.
... Tat mRNA was prepared with an Ambion mMESSAGE mMACHINE T7 ULTRA transcription kit (Thermo Fisher Scientific). The full-length HIV proviral clones pNL-Lai, 32 pNL(AD8), 33 Bal, 34 JRSCF, 35 pNL-YU2, 36 and 89.6 37 have been previously described. GFP-expressing control lentiviral vector (pTY-EFeGFP) was kindly provided by the NIH AIDS Reagent Program and packaged as described subsequently. ...
... Several HIV LTR-based Tat-and Rev-dependent lentiviral vectors have been developed by numerous groups. 8,10,24,36,42 Another important aspect of using conditional vectors is that the anti-HIV gene needs to target late stages of the virus, as Tatmediated activation in infected cells would not prevent early steps in the HIV life cycle. In this regard, we have previously described the use of 14 In this study we have used the TK-SR39 gene in a conditional Tat-dependent vector for elimination of HIV-infected cells. ...
Gene therapy remains one of the potential strategies to achieve HIV cure. One of the major limitations of anti-HIV gene therapy is recovering adequate number of modified cells to generate an HIV proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector transformed cells for selection and subsequently target HIV infected cells for elimination via treatment with Ganciclovir (GCV). We utilized the HSV Thymidine Kinase (TK) mutant SR39 that is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector pNL-GFPRRESA that expresses the gene of interest as well as GFP in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that TK-WT at eliminating infected cells at lower concentration of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either via tat RNA transfection or transduction via a non-integrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected with this approach, infection with CXCR4 tropic Lai virus could be suppressed via treatment with GCV. GCV treatment limited the number of HIV infected cells, virus production as well as virus induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy.
... We have previously demonstrated the phenomenon of bystander apoptosis mediated by HIV Env both in vitro (18,19) and in vivo (20), and found that Env fusogenic activity correlates with bystander apoptosis and CD4 decline, but not virus replication. This phenomenon is not limited to laboratory-adapted viruses but is also seen with a variety of Envs derived from HIV-infected patients (21). The high variability in the bystander apoptosis-inducing potential (AIP) of primary Envs suggests that phenotypic variability may play a role in the differential rates of disease progression. ...
... In this study, we analyzed samples from 50 HIV-infected patients for multiple immunopathological markers including those for IA and apoptosis in CD4 + and CD8 + cells. Furthermore, we cloned full-length functional env genes from 11 viremic HIV + patients and characterized the derived Env glycoproteins for their AIP using a unique assay developed in our laboratory (21). Our results demonstrate that the AIP of patient Envs correlates inversely with the CD4:CD8 ratios. ...
... Bystander AIP of cloned Envs was determined using a coculture assay described previously (21). In brief, HeLa cells transfected with the laboratory-adapted or cloned primary Env constructs were cocultured either with SupT-R5-H6 cells (27) or with resting or activated PBMCs. ...
The mechanism behind the selective depletion of CD4(+) cells in HIV infections remains undetermined. Although HIV selectively infects CD4(+) cells, the relatively few infected cells in vivo cannot account for the extent of CD4(+) T cell depletion, suggesting indirect or bystander mechanisms. The role of virus replication, Env glycoprotein phenotype, and immune activation (IA) in this bystander phenomenon remains controversial. Using samples derived from HIV-infected patients, we demonstrate that, although IA in both CD4(+) and CD8(+) subsets correlates with CD4 decline, apoptosis in CD4(+) and not CD8(+) cells is associated with disease progression. Because HIV-1 Env glycoprotein has been implicated in bystander apoptosis, we cloned full-length Envs from plasma of viremic patients and tested their apoptosis-inducing potential (AIP). Interestingly, AIP of HIV-1 Env glycoproteins were found to correlate inversely with CD4:CD8 ratios, suggesting a role of Env phenotype in disease progression. In vitro mitogenic stimulation of PBMCs resulted in upregulation of IA markers but failed to alter the CD4:CD8 ratio. However, coculture of normal PBMCs with Env-expressing cells resulted in selective CD4 loss that was significantly enhanced by IA. Our study demonstrates that AIP of HIV-1 Env and IA collectively determine CD4 loss in HIV infection.
Human immunodeficiency virus-1 (HIV-1) causes CD4 T cell depletion through a number of mechanisms, including programmed cell death pathways (both apoptotic and non-apoptotic). In the setting of HIV-1 infection, the enhanced lymphocyte cell death occurs as a consequence of complex interactions between the host immune system and viral factors, which are reviewed herein. On the other hand, the main challenge to HIV-1 eradication is the development of latent infection in a subset of long lived cells, including CD4+ T cells and macrophages, which resist HIV-induced cell death. Understanding the potential mechanisms of how HIV-1 induces lymphocyte cell death is critical to the "kick and kill" cure strategy, which relies on the effective killing of reactivated, HIV-1 infected cells.
