Himanshu Garg | Chandigarh University Gharuan (original) (raw)

Papers by Himanshu Garg

BMC Cell Biology, 2009

Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by ir... more Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.

Trends in Molecular Medicine, 2009

Silencing specific gene expression by RNA interference (RNAi) has rapidly become a standard tool ... more Silencing specific gene expression by RNA interference (RNAi) has rapidly become a standard tool for the reverse genetic analysis of gene functions. It also has tremendous potential for managing diseases for which effective treatment is currently unavailable or suboptimal. However, the poor cellular uptake of synthetic small interfering RNAs (siRNAs) is a major impediment for their clinical use. Great progress has been made in recent years to overcome this barrier, and several methods have been described for the in vivo delivery of siRNA. Moreover, the latest advances have focused on achieving targeted siRNA delivery restricted to relevant tissues and cell types in vivo. These approaches are expected to reduce the dose requirement as well as minimize siRNA-induced toxicities, thereby advancing the field of siRNA therapy towards clinical use.

Cancer Gene Therapy, 2010

Suicide gene vectors are being developed in many laboratories as an attractive approach to cancer... more Suicide gene vectors are being developed in many laboratories as an attractive approach to cancer therapy. However, the development of these therapies is hampered by safety concerns and limitations of efficacy. The use of tumor-specific promoters, such as survivin promoter, can provide much needed specificity to target tumor cells. However, the expression levels from these promoters is often suboptimal and hence it is imperative to enhance the activity of the cytotoxic gene of interest. We tested apoptotic activity of several mutants of proapoptotic gene bax that constitutively translocate to the mitochondria and induce apoptosis. One of these mutants with deletion of serine at position S184 (S184del) was found to be most active and showed significant antitumor activity when expressed by the survivin promoter. In vitro testing shows that this vector (Sur-BaxS184del) induces cell killing in a variety of tumor cell lines of different origin with significantly higher efficacy than wild-type bax (Sur-BaxWT). The increase in cytotoxicity was a result of enhanced induction of apoptosis in tumor cells. In contrast to cytomegalovirus (CMV) promoter-driven bax (CMV-Bax), Sur-BaxS184del caused minimum toxicity in normal human dermal fibroblasts validating its specificity and safety. In a mouse tumor model (DA-3, murine breast cancer cells), we show that intratumoral injection of Sur-BaxS184del resulted in tumor growth retardation to the same level as CMV-Bax. This study highlights the effectiveness of using bax mutants in combination with survivin promoter for tumor-targeted suicide gene therapy in a nonviral vector.

Virology, 2004

Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human imm... more Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU 1 failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using sitedirected mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation.

Virology, 2004

Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persist... more Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persists as both a latent and productive infection in subsets of CD4 + cells. Reports regarding establishment of a stable HIV-1 infection in quiescent T cells in vitro, however, are controversial. In the present study, we investigated the susceptibility of naive and activated CD4 + cell subsets (distinguished by differential expression of CD25) to feline immunodeficiency virus (FIV) infection, their ability to replicate the virus, and potentially act as a reservoir for virus persistence in infected animals. While both CD4 + CD25 + and CD4 + CD25 À cells are susceptible to FIV infection in vitro and in vivo, only CD4 + CD25 + cells produce infectious virions when cultured with interleukin-2 (IL-2). Latently infected CD4 + CD25 À cells produce infectious virions following ConcanvalinA (ConA) stimulation, which correlates with upregulated surface expression of CD25. In contrast to CD4 + CD25 À cells, CD4 + CD25 + cells remain unresponsive to mitogen stimulation and are relatively resistant to apoptosis whether or not infected with FIV. The ability of CD4 + CD25 + cells to replicate FIV efficiently in the presence of IL-2 but remain anergic and unresponsive to apoptotic signaling suggests that these cells may provide a reservoir of productive FIV infection. On the contrary, CD4 + CD25 À cells seem to establish as latent viral reservoirs capable of being reactivated after stimulation. D

Vaccine, 2008

Our overall goal is to understand how viral envelope proteins mediate membrane fusion and pathoge... more Our overall goal is to understand how viral envelope proteins mediate membrane fusion and pathogenesis. Membrane fusion is a crucial step in the delivery of the viral genome into the cell resulting in infection. On the other hand, fusion activity of viral envelope glycoproteins expressed in infected cells may cause the demise of uninfected bystander cells by apoptosis. Our general approach is to kinetically resolve steps in the pathway of viral envelope glycoprotein-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. Since HIV fusion involves a complex cascade of interactions of the envelope glycoprotein with two receptors, membrane organization plays an important role and interfering with it may modulate entry. To study this phenomenon, we have either examined cell lines with differential expression of sphingolipids (such as GM3), or altered membrane organization by modifying levels of cholesterol, ceramides, or glycosphingolipids. We show that the localized plasma membrane lipid microenvironment (and not the specific membrane lipids) in the vicinity of CD4 controls receptor mobility and HIV-1 fusion. The complex cascade of conformational changes that must occur to allow virus entry is also a very important target for therapy and vaccine development. We have recently designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to promote permanent attachment. Using a temperature-arrested state in vitro assay we show evidence for the trapping of a pre-six-helix bundle fusion intermediate by a covalent reaction with the inhibitory reactive peptide. Also, using photo-reactive hydrophobic probes we have found ways to inactivate viral envelope glycoproteins while leaving their overall structures intact. Finally, in order to study the envelope glycoprotein effects on pathogenesis, we have used an in vitro model of co-culture of envelope-expressing cells as effectors and CD4+ T cells as targets. We delineated that apoptosis mediated by envelope glycoprotein in bystander cells correlates with transmembrane subunit (gp41)-induced hemifusion. The apoptotic pathway initiated by this interaction involves caspase-3-dependent mitochondrial depolarization and reactive oxygen species production, which depends on the phenotype of the envelope glycoprotein associated with the virus. Taken as a whole, our studies have many different important implications for antiviral therapies and vaccine development.

Experientia, 2008

Mechanisms of HIV-mediated CD4+ T cell loss leading to immunodeficiency are amongst the most exte... more Mechanisms of HIV-mediated CD4+ T cell loss leading to immunodeficiency are amongst the most extensively studied yet unanswered questions in HIV biology. The level of CD4+ T cell depletion in HIV infected patients far exceeds the number of infected T cells, suggesting an indirect mechanism of HIV pathogenesis termed bystander cell death. Evidence is accumulating that the HIV envelope glycoprotein (Env) is a major determinant of HIV pathogenesis and plays a critical role in bystander cell death. The complex structure and function of HIV Env makes the determination of the mechanism of Envmediated apoptosis more complex than previously thought. This review will examine the complex relationship between HIV Env phenotype, coreceptor expression and immune activation in determining HIV pathogenesis. We review data here corresponding to the role of HIV Env hemifusion activity in HIV pathogenesis and how it interplays with other AIDS associated factors such as chemokine receptor expression and immune activation.

Bioorganic & Medicinal Chemistry, 2011

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of βgalactosylceram... more Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of βgalactosylceramide (GalCer), a set of mono-and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC 50 's were in the 50 μM range with no toxicity to the cells at concentrations up to 200 μM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.

Journal of Leukocyte Biology, 2005

Apoptotic loss of CD4؉ T cells has been proposed as a mechanism of T cell depletion in human immu... more Apoptotic loss of CD4؉ T cells has been proposed as a mechanism of T cell depletion in human immunodeficiency virus (HIV) infections resulting in immunodeficiency. The Env glycoprotein has been implicated in apoptosis of uninfected bystander cells via gp120 binding to CD4/CXC chemokine receptor 4 as well as the fusion/hemifusion process mediated by gp41.

Virology, 2005

Cellular activation plays an important role in retroviral replication. Previously, we have shown ... more Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4(+)CD25(+) T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4(+)CD25(+) and CD4(+)CD25(-) cells under different stimulation conditions. Both CD4(+)CD25(+) and CD4(+)CD25(-) cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4(+)CD25(+) cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4(+)CD25(-) T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4(+)CD25(-) cells to replicate FIV, it induces apoptosis in a high percentage of CD4(+)CD25(+) T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4(+)CD25(-) cells. These results suggest that CD4(+)CD25(+) and CD4(+)CD25(-) T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4(+)CD25(+) and CD4(+)CD25(-) cells to serve as potential reservoirs of a productive and latent FIV infection.

Virology Journal, 2011

The mechanism by which HIV infection leads to a selective depletion of CD4 cells leading to immun... more The mechanism by which HIV infection leads to a selective depletion of CD4 cells leading to immunodeficiency remains highly debated. Whether the loss of CD4 cells is a direct consequence of virus infection or bystander apoptosis of uninfected cells is also uncertain.

Virology, 2004

Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which p... more Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function. D

Retrovirology, 2006

Background: HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescenc... more Background: HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescence of the HIV gp41 N-helical and C-helical regions, which results in the formation of 6 helix bundles. Kinetics of HIV Env-mediated fusion is an important determinant of sensitivity to entry inhibitors and antibodies. However, the parameters that govern the HIV Env fusion cascade have yet to be fully elucidated. We address this issue by comparing the kinetics HIV-1 IIIB Env with those mediated by HIV-2 from two strains with different affinities for CD4 and CXCR4.

Biophysical Journal, 2010

Aids Research and Human Retroviruses, 2009

In previous studies on mechanisms of HIV-1-mediated pathogenesis we showed that bystander apoptos... more In previous studies on mechanisms of HIV-1-mediated pathogenesis we showed that bystander apoptosis mediated by cell surface-expressed HIV-1 Env correlated with the fusogenic properties of the gp41 subunit of Env. A crucial step in HIV gp41-mediated fusion is the refolding of the protein into a six-helix bundle along the N- and C-terminal coiled-coil domains. These domains have been targeted by peptide inhibitors that inhibit gp41-mediated fusion. One of these inhibitors, enfuvirtide, is the first such drug approved for therapy. More recently, clinical data suggest that the beneficial effects of enfuvirtide extend beyond virus suppression and are associated with certain resistance mutations in gp41. In this study we characterized the bystander apoptosis-inducing potential of mutants associated with increased CD4 counts that arise during enfuvirtide therapy. Whereas all mutant clones were reduced in both cell-to-cell fusion activity and apoptosis induction there was limited effect on virus infection or replication. The viruses were found to have apoptosis-inducing activity in the order WT > V38M > V38A > G36D > V38E, which correlated with cell-to-cell fusion but not infection. Interestingly, the level of resistance as determined by the IC(50) of enfuvirtide also correlated inversely with both cell fusion and apoptosis in that the most resistant Envs were the least fusogenic and pathogenic. This suggests the beneficial effects of enfuvirtide therapy beyond virus suppression may be mediated by selecting less pathogenic HIV isolates over time.

Virology, 2005

Cellular activation plays an important role in retroviral replication. Previously, we have shown ... more Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4(+)CD25(+) T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4(+)CD25(+) and CD4(+)CD25(-) cells under different stimulation conditions. Both CD4(+)CD25(+) and CD4(+)CD25(-) cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4(+)CD25(+) cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4(+)CD25(-) T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4(+)CD25(-) cells to replicate FIV, it induces apoptosis in a high percentage of CD4(+)CD25(+) T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4(+)CD25(-) cells. These results suggest that CD4(+)CD25(+) and CD4(+)CD25(-) T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4(+)CD25(+) and CD4(+)CD25(-) cells to serve as potential reservoirs of a productive and latent FIV infection.

Journal of Virology, 2005

BMC Cell Biology, 2009

Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by ir... more Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.

Trends in Molecular Medicine, 2009

Silencing specific gene expression by RNA interference (RNAi) has rapidly become a standard tool ... more Silencing specific gene expression by RNA interference (RNAi) has rapidly become a standard tool for the reverse genetic analysis of gene functions. It also has tremendous potential for managing diseases for which effective treatment is currently unavailable or suboptimal. However, the poor cellular uptake of synthetic small interfering RNAs (siRNAs) is a major impediment for their clinical use. Great progress has been made in recent years to overcome this barrier, and several methods have been described for the in vivo delivery of siRNA. Moreover, the latest advances have focused on achieving targeted siRNA delivery restricted to relevant tissues and cell types in vivo. These approaches are expected to reduce the dose requirement as well as minimize siRNA-induced toxicities, thereby advancing the field of siRNA therapy towards clinical use.

Cancer Gene Therapy, 2010

Suicide gene vectors are being developed in many laboratories as an attractive approach to cancer... more Suicide gene vectors are being developed in many laboratories as an attractive approach to cancer therapy. However, the development of these therapies is hampered by safety concerns and limitations of efficacy. The use of tumor-specific promoters, such as survivin promoter, can provide much needed specificity to target tumor cells. However, the expression levels from these promoters is often suboptimal and hence it is imperative to enhance the activity of the cytotoxic gene of interest. We tested apoptotic activity of several mutants of proapoptotic gene bax that constitutively translocate to the mitochondria and induce apoptosis. One of these mutants with deletion of serine at position S184 (S184del) was found to be most active and showed significant antitumor activity when expressed by the survivin promoter. In vitro testing shows that this vector (Sur-BaxS184del) induces cell killing in a variety of tumor cell lines of different origin with significantly higher efficacy than wild-type bax (Sur-BaxWT). The increase in cytotoxicity was a result of enhanced induction of apoptosis in tumor cells. In contrast to cytomegalovirus (CMV) promoter-driven bax (CMV-Bax), Sur-BaxS184del caused minimum toxicity in normal human dermal fibroblasts validating its specificity and safety. In a mouse tumor model (DA-3, murine breast cancer cells), we show that intratumoral injection of Sur-BaxS184del resulted in tumor growth retardation to the same level as CMV-Bax. This study highlights the effectiveness of using bax mutants in combination with survivin promoter for tumor-targeted suicide gene therapy in a nonviral vector.

Virology, 2004

Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human imm... more Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU 1 failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using sitedirected mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation.

Virology, 2004

Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persist... more Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persists as both a latent and productive infection in subsets of CD4 + cells. Reports regarding establishment of a stable HIV-1 infection in quiescent T cells in vitro, however, are controversial. In the present study, we investigated the susceptibility of naive and activated CD4 + cell subsets (distinguished by differential expression of CD25) to feline immunodeficiency virus (FIV) infection, their ability to replicate the virus, and potentially act as a reservoir for virus persistence in infected animals. While both CD4 + CD25 + and CD4 + CD25 À cells are susceptible to FIV infection in vitro and in vivo, only CD4 + CD25 + cells produce infectious virions when cultured with interleukin-2 (IL-2). Latently infected CD4 + CD25 À cells produce infectious virions following ConcanvalinA (ConA) stimulation, which correlates with upregulated surface expression of CD25. In contrast to CD4 + CD25 À cells, CD4 + CD25 + cells remain unresponsive to mitogen stimulation and are relatively resistant to apoptosis whether or not infected with FIV. The ability of CD4 + CD25 + cells to replicate FIV efficiently in the presence of IL-2 but remain anergic and unresponsive to apoptotic signaling suggests that these cells may provide a reservoir of productive FIV infection. On the contrary, CD4 + CD25 À cells seem to establish as latent viral reservoirs capable of being reactivated after stimulation. D

Vaccine, 2008

Our overall goal is to understand how viral envelope proteins mediate membrane fusion and pathoge... more Our overall goal is to understand how viral envelope proteins mediate membrane fusion and pathogenesis. Membrane fusion is a crucial step in the delivery of the viral genome into the cell resulting in infection. On the other hand, fusion activity of viral envelope glycoproteins expressed in infected cells may cause the demise of uninfected bystander cells by apoptosis. Our general approach is to kinetically resolve steps in the pathway of viral envelope glycoprotein-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. Since HIV fusion involves a complex cascade of interactions of the envelope glycoprotein with two receptors, membrane organization plays an important role and interfering with it may modulate entry. To study this phenomenon, we have either examined cell lines with differential expression of sphingolipids (such as GM3), or altered membrane organization by modifying levels of cholesterol, ceramides, or glycosphingolipids. We show that the localized plasma membrane lipid microenvironment (and not the specific membrane lipids) in the vicinity of CD4 controls receptor mobility and HIV-1 fusion. The complex cascade of conformational changes that must occur to allow virus entry is also a very important target for therapy and vaccine development. We have recently designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to promote permanent attachment. Using a temperature-arrested state in vitro assay we show evidence for the trapping of a pre-six-helix bundle fusion intermediate by a covalent reaction with the inhibitory reactive peptide. Also, using photo-reactive hydrophobic probes we have found ways to inactivate viral envelope glycoproteins while leaving their overall structures intact. Finally, in order to study the envelope glycoprotein effects on pathogenesis, we have used an in vitro model of co-culture of envelope-expressing cells as effectors and CD4+ T cells as targets. We delineated that apoptosis mediated by envelope glycoprotein in bystander cells correlates with transmembrane subunit (gp41)-induced hemifusion. The apoptotic pathway initiated by this interaction involves caspase-3-dependent mitochondrial depolarization and reactive oxygen species production, which depends on the phenotype of the envelope glycoprotein associated with the virus. Taken as a whole, our studies have many different important implications for antiviral therapies and vaccine development.

Experientia, 2008

Mechanisms of HIV-mediated CD4+ T cell loss leading to immunodeficiency are amongst the most exte... more Mechanisms of HIV-mediated CD4+ T cell loss leading to immunodeficiency are amongst the most extensively studied yet unanswered questions in HIV biology. The level of CD4+ T cell depletion in HIV infected patients far exceeds the number of infected T cells, suggesting an indirect mechanism of HIV pathogenesis termed bystander cell death. Evidence is accumulating that the HIV envelope glycoprotein (Env) is a major determinant of HIV pathogenesis and plays a critical role in bystander cell death. The complex structure and function of HIV Env makes the determination of the mechanism of Envmediated apoptosis more complex than previously thought. This review will examine the complex relationship between HIV Env phenotype, coreceptor expression and immune activation in determining HIV pathogenesis. We review data here corresponding to the role of HIV Env hemifusion activity in HIV pathogenesis and how it interplays with other AIDS associated factors such as chemokine receptor expression and immune activation.

Bioorganic & Medicinal Chemistry, 2011

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of βgalactosylceram... more Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of βgalactosylceramide (GalCer), a set of mono-and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC 50 's were in the 50 μM range with no toxicity to the cells at concentrations up to 200 μM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.

Journal of Leukocyte Biology, 2005

Apoptotic loss of CD4؉ T cells has been proposed as a mechanism of T cell depletion in human immu... more Apoptotic loss of CD4؉ T cells has been proposed as a mechanism of T cell depletion in human immunodeficiency virus (HIV) infections resulting in immunodeficiency. The Env glycoprotein has been implicated in apoptosis of uninfected bystander cells via gp120 binding to CD4/CXC chemokine receptor 4 as well as the fusion/hemifusion process mediated by gp41.

Virology, 2005

Cellular activation plays an important role in retroviral replication. Previously, we have shown ... more Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4(+)CD25(+) T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4(+)CD25(+) and CD4(+)CD25(-) cells under different stimulation conditions. Both CD4(+)CD25(+) and CD4(+)CD25(-) cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4(+)CD25(+) cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4(+)CD25(-) T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4(+)CD25(-) cells to replicate FIV, it induces apoptosis in a high percentage of CD4(+)CD25(+) T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4(+)CD25(-) cells. These results suggest that CD4(+)CD25(+) and CD4(+)CD25(-) T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4(+)CD25(+) and CD4(+)CD25(-) cells to serve as potential reservoirs of a productive and latent FIV infection.

Virology Journal, 2011

The mechanism by which HIV infection leads to a selective depletion of CD4 cells leading to immun... more The mechanism by which HIV infection leads to a selective depletion of CD4 cells leading to immunodeficiency remains highly debated. Whether the loss of CD4 cells is a direct consequence of virus infection or bystander apoptosis of uninfected cells is also uncertain.

Virology, 2004

Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which p... more Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function. D

Retrovirology, 2006

Background: HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescenc... more Background: HIV envelope glycoprotein (Env)-mediated fusion is driven by the concerted coalescence of the HIV gp41 N-helical and C-helical regions, which results in the formation of 6 helix bundles. Kinetics of HIV Env-mediated fusion is an important determinant of sensitivity to entry inhibitors and antibodies. However, the parameters that govern the HIV Env fusion cascade have yet to be fully elucidated. We address this issue by comparing the kinetics HIV-1 IIIB Env with those mediated by HIV-2 from two strains with different affinities for CD4 and CXCR4.

Biophysical Journal, 2010

Aids Research and Human Retroviruses, 2009

In previous studies on mechanisms of HIV-1-mediated pathogenesis we showed that bystander apoptos... more In previous studies on mechanisms of HIV-1-mediated pathogenesis we showed that bystander apoptosis mediated by cell surface-expressed HIV-1 Env correlated with the fusogenic properties of the gp41 subunit of Env. A crucial step in HIV gp41-mediated fusion is the refolding of the protein into a six-helix bundle along the N- and C-terminal coiled-coil domains. These domains have been targeted by peptide inhibitors that inhibit gp41-mediated fusion. One of these inhibitors, enfuvirtide, is the first such drug approved for therapy. More recently, clinical data suggest that the beneficial effects of enfuvirtide extend beyond virus suppression and are associated with certain resistance mutations in gp41. In this study we characterized the bystander apoptosis-inducing potential of mutants associated with increased CD4 counts that arise during enfuvirtide therapy. Whereas all mutant clones were reduced in both cell-to-cell fusion activity and apoptosis induction there was limited effect on virus infection or replication. The viruses were found to have apoptosis-inducing activity in the order WT > V38M > V38A > G36D > V38E, which correlated with cell-to-cell fusion but not infection. Interestingly, the level of resistance as determined by the IC(50) of enfuvirtide also correlated inversely with both cell fusion and apoptosis in that the most resistant Envs were the least fusogenic and pathogenic. This suggests the beneficial effects of enfuvirtide therapy beyond virus suppression may be mediated by selecting less pathogenic HIV isolates over time.

Virology, 2005

Cellular activation plays an important role in retroviral replication. Previously, we have shown ... more Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4(+)CD25(+) T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4(+)CD25(+) and CD4(+)CD25(-) cells under different stimulation conditions. Both CD4(+)CD25(+) and CD4(+)CD25(-) cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4(+)CD25(+) cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4(+)CD25(-) T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4(+)CD25(-) cells to replicate FIV, it induces apoptosis in a high percentage of CD4(+)CD25(+) T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4(+)CD25(-) cells. These results suggest that CD4(+)CD25(+) and CD4(+)CD25(-) T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4(+)CD25(+) and CD4(+)CD25(-) cells to serve as potential reservoirs of a productive and latent FIV infection.

Journal of Virology, 2005