Michael Roche

Publications of Michael Roche

• HIV-1 predisposed to acquiring resistance to maraviroc (MVC) and other CCR5 antagonists in vitro has an inherent, low-level ability to utilize MVC-bound CCR5 for entry.

  Authors: Michael Roche, Martin R Jakobsen, Anne Ellett, Hamid Salimiseyedabad, Becky Jubb, Mike Westby, Benhur Lee, Sharon R Lewin, Melissa J Churchill, Paul R Gorry

  Retrovirology. 11/2011; 8:89.

  Maraviroc (MVC) and other CCR5 antagonists are HIV-1 entry inhibitors that bind to- and alter the conformation of CCR5, such that CCR5 is no longer recognized by the viral gp120 envelope (Env)Maraviroc (MVC) and other CCR5 antagonists are HIV-1 entry inhibitors that bind to- and alter the conformation of CCR5, such that CCR5 is no longer recognized by the viral gp120 envelope (Env) glycoproteins. Resistance to CCR5 antagonists results from HIV-1 Env acquiring the ability to utilize the drug-bound conformation of CCR5. Selecting for HIV-1 resistance to CCR5-antagonists in vitro is relatively difficult. However, the CCR5-using CC1/85 strain appears to be uniquely predisposed to acquiring resistance to several CCR5 antagonists in vitro including MVC, vicriviroc and AD101. Here, we show that Env derived from the parental CC1/85 strain is inherently capable of a low affinity interaction with MVC-bound CCR5. However, this phenotype was only revealed in 293-Affinofile cells and NP2-CD4/CCR5 cells that express very high levels of CCR5, and was masked in TZM-bl, JC53 and U87-CD4/CCR5 cells as well as PBMC, which express comparatively lower levels of CCR5 and which are more commonly used to detect resistance to CCR5 antagonists. Env derived from the CC1/85 strain of HIV-1 is inherently capable of a low-affinity interaction with MVC-bound CCR5, which helps explain the relative ease in which CC1/85 can acquire resistance to CCR5 antagonists in vitro. The detection of similar phenotypes in patients may identify those who could be at higher risk of virological failure on MVC.

• Alternative coreceptor requirements for efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.

  Authors: Kieran Cashin, Michael Roche, Jasminka Sterjovski, Anne Ellett, Lachlan R Gray, Anthony L Cunningham, Paul A Ramsland, Melissa J Churchill, Paul R Gorry

  Journal of virology. 08/2011; 85(20):10699-709.

  Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute toMacrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.

• CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity.

  Authors: Jasminka Sterjovski, Melissa J Churchill, Michael Roche, Anne Ellett, William Farrugia, Steven L Wesselingh, Anthony L Cunningham, Paul A Ramsland, Paul R Gorry

  Virology. 02/2011; 410(2):418-28.

  CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, weCD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n=16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

• HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism.

  Authors: Michael Roche, Martin R Jakobsen, Jasminka Sterjovski, Anne Ellett, Filippo Posta, Benhur Lee, Becky Jubb, Mike Westby, Sharon R Lewin, Paul A Ramsland, Melissa J Churchill, Paul R Gorry

  Journal of virology. 02/2011; 85(9):4330-42.

  Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results fromMaraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.

• An altered and more efficient mechanism of CCR5 engagement contributes to macrophage tropism of CCR5-using HIV-1 envelopes.

  Authors: Jasminka Sterjovski, Michael Roche, Melissa J Churchill, Anne Ellett, William Farrugia, Lachlan R Gray, Daniel Cowley, Pantelis Poumbourios, Benhur Lee, Steven L Wesselingh, Anthony L Cunningham, Paul A Ramsland, Paul R Gorry

  Virology. 09/2010; 404(2):269-78.

  While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env)While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env) clones to characterize virus-cell interactions important for efficient CCR5-mediated macrophage entry. The magnitude of macrophage entry by Env-pseudotyped reporter viruses correlated with increased immunoreactivity of CD4-induced gp120 epitopes, increased ability to scavenge low levels of cell-surface CCR5, reduced sensitivity to the CCR5 inhibitor maraviroc, and increased dependence on specific residues in the CCR5 ECL2 region. These results are consistent with an altered and more efficient mechanism of CCR5 engagement. Structural studies revealed potential alterations within the gp120 V3 loop, the gp41 interaction sites at the gp120 C- and N-termini, and within the gp120 CD4 binding site which may directly or indirectly lead to more efficient CCR5-usage. Thus, enhanced gp120-CCR5 interactions may contribute to M-tropism of R5 HIV-1 strains through different structural mechanisms.

• X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

  Authors: Kate L Jones, Michael Roche, Michael P Gantier, Nasim A Begum, Tasuku Honjo, Salvatore Caradonna, Bryan R G Williams, Johnson Mak

  The Journal of biological chemistry. 04/2010; 285(24):18603-14.

  It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinctIt has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations.

• Enhanced CD4+ cellular apoptosis by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with progressive HIV-1 infection.

  Authors: Jessica Wade, Jasminka Sterjovski, Lachlan Gray, Michael Roche, Lisa Chiavaroli, Anne Ellett, Martin R Jakobsen, Daniel Cowley, Candida da Fonseca Pereira, Nitin Saksena, Bin Wang, Damian F J Purcell, Ingrid Karlsson, Eva Maria Fenyö, Melissa Churchill, Paul R Gorry

  Virology. 11/2009;

  CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood.CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood. We investigated mechanisms contributing to R5 envelope glycoprotein (Env)-mediated cellular apoptosis by constructing a panel of retroviral vectors engineered to co-express GFP and R5 Envs derived from two HIV-1-infected subjects spanning asymptomatic (Early, E-R5 Envs) to late stages of infection (Late, L-R5 Envs). The L-R5 Envs induced significantly more cellular apoptosis than E-R5 Envs, but only in Env-expressing (GFP-positive) cells, and only in cells where CD4 and CCR5 levels were limiting. Studies with fusion-defective Env mutants showed induction of apoptosis required membrane-fusing events. Our results provide evidence for an intracellular mechanism of R5 Env-induced apoptosis of CD4+ cells that requires membrane fusion. Furthermore, they contribute to a better understanding of mechanisms involved in CD4+ T-cell loss in subjects experiencing progressive R5 HIV-1 infection.

• Tissue-Specific Sequence Alterations in the HIV-1 Envelope Favoring CCR5-Usage contribute to Persistence of Dual-Tropic Virus in Brain.

  Authors: Lachlan Gray, Michael Roche, Melissa J Churchill, Jasminka Sterjovski, Anne Ellett, Pantelis Poumbourios, Shameem Sheffief, Bin Wang, Nitin Saksena, Damian F J Purcell, Steven Wesselingh, Anthony L Cunningham, Bruce J Brew, Dana Gabuzda, Paul R Gorry

  Journal of virology. 04/2009;

  Most human immunodeficiency virus type 1 (HIV-1) strains isolated from brain use CCR5 for entry into macrophages and microglia. Strains that use both CCR5 and CXCR4 for entry (R5X4 strains) have beenMost human immunodeficiency virus type 1 (HIV-1) strains isolated from brain use CCR5 for entry into macrophages and microglia. Strains that use both CCR5 and CXCR4 for entry (R5X4 strains) have been identified in brain of some individuals, but mechanisms underlying the persistence of R5X4 viruses compartmentalized between brain and other tissue reservoirs are unknown. Here, we characterized changes in the HIV-1 envelope (Env) that enhance the tropism of R5X4 variants for brain or lymphoid tissue. R5X4 Envs derived from brain of two individuals had enhanced CCR5-usage in fusion assays compared to R5X4 Envs derived from matched spleen or blood, which was associated with reduced dependence on specific residues in the CCR5 N-terminus and extracellular loop (ECL)1 and ECL3 regions. In contrast, spleen/blood-derived Envs had enhanced CXCR4-usage compared to brain-derived Envs, which was associated with reduced dependence on residues in the CXCR4 N-terminus and ECL2 region. Consequently, brain-derived Envs had preferential CCR5-usage for HIV-1 entry into the JC53 cell line, could use either CCR5 or CXCR4 for entry into monocyte-derived macrophages (MDM) and could use CCR5 (albeit inefficiently) for entry into peripheral blood mononuclear cells (PBMC), whereas the entry of spleen-derived Envs was CXCR4-dependent in all three cell types. Mutagenesis studies on Env amino acid variants influencing coreceptor usage showed S306 in the gp120 V3 region of brain-derived Envs reduces dependence on the CCR5 N-terminus and enhances CCR5-usage for HIV-1 entry into PBMC and MDM, whereas R306 in spleen-derived Envs reduces dependence on the CXCR4-N-terminus and confers the CXCR4-restricted phenotype. These results identify mechanisms underlying R5X4 HIV-1 persistence in different tissue reservoirs. Tissue-specific changes in the gp120 V3 region that increase the efficiency of CCR5 or CXCR4 usage, and thereby influence coreceptor preference, may enhance the tropism of R5X4 strains for CCR5-expressing macrophage-lineage cells in brain and CXCR4-expressing T-cells in lymphoid tissues, respectively.

• Pathogenicity and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo.

  Authors: Paul R Gorry, Dale A McPhee, Erin Verity, Wayne B Dyer, Steven L Wesselingh, Jennifer Learmont, John S Sullivan, Michael Roche, John J Zaunders, Dana Gabuzda, Suzanne M Crowe, John Mills, Sharon R Lewin, Bruce J Brew, Anthony L Cunningham, Melissa J Churchill

  Retrovirology. 02/2007; 4:66.

  In efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef and otherIn efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef and other accessory genes. Although live attenuated HIV vaccines are unlikely to be developed due to a myriad of safety concerns, opportunities exist to better understand the correlates of immune protection against HIV infection by studying rare cohorts of long-term survivors infected with attenuated, nef-deleted HIV strains such as the Sydney blood bank cohort (SBBC). Here, we review studies of viral evolution, pathogenicity, and immune responses to HIV infection in SBBC members. The studies show that potent, broadly neutralizing anti-HIV antibodies and robust CD8+ T-cell responses to HIV infection were not necessary for long-term control of HIV infection in a subset of SBBC members, and were not sufficient to prevent HIV sequence evolution, augmentation of pathogenicity and eventual progression of HIV infection in another subset. However, a persistent T-helper proliferative response to HIV p24 antigen was associated with long-term control of infection. Together, these results underscore the importance of the host in the eventual outcome of infection. Thus, whilst generating an effective antibody and CD8+ T-cell response are an essential component of vaccines aimed at preventing primary HIV infection, T-helper responses may be important in the generation of an effective therapeutic vaccine aimed at blunting chronic HIV infection.

• Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

  Authors: Jasminka Sterjovski, Melissa Churchill, Anne Ellett, Lachlan Gray, Michael Roche, Rebecca Dunfee, Damian Purcell, Nitin Saksena, Bin Wang, Secondo Sonza, Steven Wesselingh, Ingrid Karlsson, Eva-Maria Fenyo, Dana Gabuzda, Anthony Cunningham, Paul Gorry

  Retrovirology. 01/2007;

  Abstract Background CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlyingAbstract Background CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively. Results Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. Conclusion Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.

• Pathogenicity and immunogenicity of attenuated, nef -deleted HIV-1 strains in vivo

  Authors: Paul Gorry, Dale McPhee, Erin Verity, Wayne Dyer, Steven Wesselingh, Jennifer Learmont, John Sullivan, Michael Roche, John Zaunders, Dana Gabuzda, Suzanne Crowe, John Mills, Sharon Lewin, Bruce Brew, Anthony Cunningham, Melissa Churchill

  Retrovirology. 01/2007;

  Abstract In efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef andAbstract In efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef and other accessory genes. Although live attenuated HIV vaccines are unlikely to be developed due to a myriad of safety concerns, opportunities exist to better understand the correlates of immune protection against HIV infection by studying rare cohorts of long-term survivors infected with attenuated, nef -deleted HIV strains such as the Sydney blood bank cohort (SBBC). Here, we review studies of viral evolution, pathogenicity, and immune responses to HIV infection in SBBC members. The studies show that potent, broadly neutralizing anti-HIV antibodies and robust CD8+ T-cell responses to HIV infection were not necessary for long-term control of HIV infection in a subset of SBBC members, and were not sufficient to prevent HIV sequence evolution, augmentation of pathogenicity and eventual progression of HIV infection in another subset. However, a persistent T-helper proliferative response to HIV p24 antigen was associated with long-term control of infection. Together, these results underscore the importance of the host in the eventual outcome of infection. Thus, whilst generating an effective antibody and CD8+ T-cell response are an essential component of vaccines aimed at preventing primary HIV infection, T-helper responses may be important in the generation of an effective therapeutic vaccine aimed at blunting chronic HIV infection.

• CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120–CD4 interactions and fusogenicity

  Authors: Jasminka Sterjovski, Melissa J. Churchill, Michael Roche, Anne Ellett, William Farrugia, Steven L. Wesselingh, Anthony L. Cunningham, Paul A. Ramsland, Paul R. Gorry

  Virology.

  CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, weCD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120–CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120–CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120–CD4 interactions and Env fusogenicity.

• Enhanced CD4+ cellular apoptosis by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with progressive HIV-1 infection

  Authors: Jessica Wade, Jasminka Sterjovski, Lachlan Gray, Michael Roche, Lisa Chiavaroli, Anne Ellett, Martin R. Jakobsen, Daniel Cowley, Candida da Fonseca Pereira, Nitin Saksena, Bin Wang, Damian F.J. Purcell, Ingrid Karlsson, Eva-Maria Fenyö, Melissa Churchill, Paul R. Gorry

  Virology.

  CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood.CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood. We investigated mechanisms contributing to R5 envelope glycoprotein (Env)-mediated cellular apoptosis by constructing a panel of retroviral vectors engineered to co-express GFP and R5 Envs derived from two HIV-1-infected subjects spanning asymptomatic (Early, E-R5 Envs) to late stages of infection (Late, L-R5 Envs). The L-R5 Envs induced significantly more cellular apoptosis than E-R5 Envs, but only in Env-expressing (GFP-positive) cells, and only in cells where CD4 and CCR5 levels were limiting. Studies with fusion-defective Env mutants showed induction of apoptosis required membrane-fusing events. Our results provide evidence for an intracellular mechanism of R5 Env-induced apoptosis of CD4+ cells that requires membrane fusion. Furthermore, they contribute to a better understanding of mechanisms involved in CD4+ T-cell loss in subjects experiencing progressive R5 HIV-1 infection.

• An altered and more efficient mechanism of CCR5 engagement contributes to macrophage tropism of CCR5-using HIV-1 envelopes

  Authors: Jasminka Sterjovski, Michael Roche, Melissa J. Churchill, Anne Ellett, William Farrugia, Lachlan R. Gray, Daniel Cowley, Pantelis Poumbourios, Benhur Lee, Steven L. Wesselingh, Anthony L. Cunningham, Paul A. Ramsland, Paul R. Gorry

  Virology.

  While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env)While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env) clones to characterize virus-cell interactions important for efficient CCR5-mediated macrophage entry. The magnitude of macrophage entry by Env-pseudotyped reporter viruses correlated with increased immunoreactivity of CD4-induced gp120 epitopes, increased ability to scavenge low levels of cell-surface CCR5, reduced sensitivity to the CCR5 inhibitor maraviroc, and increased dependence on specific residues in the CCR5 ECL2 region. These results are consistent with an altered and more efficient mechanism of CCR5 engagement. Structural studies revealed potential alterations within the gp120 V3 loop, the gp41 interaction sites at the gp120 C- and N-termini, and within the gp120 CD4 binding site which may directly or indirectly lead to more efficient CCR5-usage. Thus, enhanced gp120-CCR5 interactions may contribute to M-tropism of R5 HIV-1 strains through different structural mechanisms.

• Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain

  Authors: Lachlan Gray, Michael Roche, Melissa J Churchill, Jasminka Sterjovski, Anne Ellett, Pantelis Poumbourios, Shameem Sherieff, Bin Wang, Nitin Saksena, Damian F J Purcell, Steven Wesselingh, Anthony L Cunningham, Bruce J Brew, Dana Gabuzda, Paul R Gorry