Quantifying the dynamics of viral recombination during free virus and cell-to-cell transmission in HIV-1 infection
Recombination has been shown to contribute to human immunodeficiency virus-1 (HIV-1) evolution in vivo, but the underlying dynamics are extremely complex, depending on the nature of the fitness landscapes and of epistatic interactions. A less well-studied determinant of recombinant evolution is the mode of virus transmission in the cell population. HIV-1 can spread by free virus transmission, resulting largely in singly infected cells, and also by direct cell-to-cell transmission, resulting in the simultaneous infection of cells with multiple viruses. We investigate the contribution of these two transmission pathways to recombinant evolution, by applying mathematical models to in vitro experimental data on the growth of fluorescent reporter viruses under static conditions (where both transmission pathways operate), and under gentle shaking conditions, where cell-to-cell transmission is largely inhibited. The parameterized mathematical models are then used to extrapolate the viral evolutionary dynamics beyond the experimental settings. Assuming a fixed basic reproductive ratio of the virus (independent of transmission pathway), we find that recombinant evolution is fastest if virus spread is driven only by cell-to-cell transmission and slows down if both transmission pathways operate. Recombinant evolution is slowest if all virus spread occurs through free virus transmission. This is due to cell-to-cell transmission 1, increasing infection multiplicity; 2, promoting the co-transmission of different virus strains from cell to cell; and 3, increasing the rate at which point mutations are generated as a result of more reverse transcription events. This study further resulted in the estimation of various parameters that characterize these evolutionary processes. For example, we estimate that during cell-to-cell transmission, an average of three viruses successfully integrated into the target cell, which can significantly raise the infection multiplicity compared to free virus transmission. In general, our study points towards the importance of infection multiplicity and cell-to-cell transmission for HIV evolution.
PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells
P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.
Mechanisms of PSGL-1 and CD43 restriction of HIV infection of CD4 T cells [Meeting Abstract]
Background: PSGL-1 (P-selectin glycoprotein ligand-1) and CD43 are surface glycoproteins that are expressed on blood CD4 T cells to bind to selectins for T cell tethering, rolling, and migration into inflamed tissues. PSGL-1 is primarily expressed on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of the endothelium for migration into inflamed tissues. Recently, PSGL-1 has also been identified as an INF-gamma-regulated anti-HIV-1 restriction factor that inactivates virion infectivity. However, the mechanisms of PSGL-1-mediated anti-HIV activity remain to be elucidated.
Method(s): We studied PSGL-1 and CD43 restriction of HIV-1 virion infectivity by co-expression of PSGL-1 or CD43 DNA with HIV-1 DNA in virion producer cells, and then quantified virion infectivity in an HIV Rev-dependent GFP indicator cell. We also studied virion incorporation of PSGL-1 by gradient ultracentrifugation and western blot detection of PSGL-1 in virion particels. In addition, we examined virion proteins of PSGL-1 imprinted particles. We also performed mapping studies to identify functional domains of PSGL-1 necessary for blocking virion infectivity. Furthermore, we performed HIV-1 entry and attachment assays to study the interaction of PSGL-1 imprinted virion particles with target cells.
Result(s): We found that the expression of PSGL-1 in virus-producing cells inhibits virion infectivity by inhibiting virion attachment to target cells. Mapping studies show that the extracellular, N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1 related monomeric E-selectin binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, downregulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally we found that PSGL-1 inhibits the infectivity of other viruses such as murine leukemia virus and influenza A virus.
Conclusion(s): These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a novel mechanism of action. Further elucidation of PSGL-1 and CD43 interaction with HIV-1 and other viruses may offer new therapeutic strategies for targeting viral infections
NNRTI-induced HIV-1 protease-mediated cytotoxicity induces rapid death of CD4 T cells during productive infection and latency reversal
BACKGROUND:Current efforts towards HIV-1 eradication focus on the reactivation and elimination of the latent viral reservoir, so-called shock and kill therapy. However, work from several groups indicates that infected cell death following virus reactivation is not guaranteed. Thus, it is imperative to develop strategies to foster specific elimination of cells carrying integrated proviruses. It has been shown that some non-nucleoside reverse transcriptaseÂ inhibitors (NNRTIs) including efavirenz can induce premature HIV-1 GagPol dimerization in productively infected cells, resulting in intracellular HIV-1 Protease (PR) activation and a reduction in HIV-1 expressing cells. RESULTS:Here, we document that NNRTI-induced PR activation triggers apoptotic death of productively infected resting or activated T cells in as little as 2Â h via caspase-dependent and independent pathways. Rilpivirine, efavirenz and etravirine were the most potent NNRTIs, whereas nevirapine had almost no effect. NNRTI-induced cell killing was prevented by inhibitors of HIV-1 Protease (PR) activity including indinavir and nelfinavir. HIV-1 transmitter founder viruses induced cell killing similarly to lab-adapted HIV-1 except when NNRTI resistance conferring mutations were present in reverse transcriptase. Mutations in PR that confer PR inhibitor (PI) resistance restore NNRTI-induced killing in the presence of PI. Finally, we show that NNRTIs can rapidly eliminate cells in which latent viruses are stimulated to active expression. CONCLUSIONS:This work supports the notion that select NNRTIs might help promote the elimination of HIV-1 producing cells as an adjuvant during shock and kill therapy.
Multiple infection of cells changes the dynamics of basic viral evolutionary processes
The infection of cells by multiple copies of a given virus can impact viral evolution in a variety of ways, yet some of the most basic evolutionary dynamics remain underexplored. Using computational models, we investigate how infection multiplicity affects the fixation probability of mutants, the rate of mutant generation, and the timing of mutant invasion. An important insight from these models is that for neutral and disadvantageous phenotypes, rare mutants initially enjoy a fitness advantage in the presence of multiple infection of cells. This arises because multiple infection allows the rare mutant to enter more target cells and to spread faster, while it does not accelerate the spread of the resident wild-type virus. The rare mutant population can increase by entry into both uninfected and wild-type-infected cells, while the established wild-type population can initially only grow through entry into uninfected cells. Following this initial advantageous phase, the dynamics are governed by drift or negative selection, respectively, and a higher multiplicity reduces the chances that mutants fix in the population. Hence, while increased infection multiplicity promotes the presence of neutral and disadvantageous mutants in the short-term, it makes it less likely in the longer term. We show how these theoretical insights can be useful for the interpretation of experimental data on virus evolution at low and high multiplicities. The dynamics explored here provide a basis for the investigation of more complex viral evolutionary processes, including recombination, reassortment, as well as complementary/inhibitory interactions.
Cofilin hyperactivation in HIV infection and targeting the cofilin pathway using an anti-Î±4Î²7 integrin antibody
A functional HIV cure requires immune reconstitution for lasting viremia control. A major immune dysfunction persisting in HIV infection is the impairment of T helper cell migration and homing to lymphoid tissues such as GALTs (gut-associated lymphoid tissues). ART (antiretroviral therapy) does not fully restore T cell motility for tissue repopulation. The molecular mechanism dictating this persistent T cell dysfunction is not understood. Cofilin is an actin-depolymerizing factor that regulates actin dynamics for T cell migration. Here, we demonstrate that blood CD4 T cells from HIV-infected patients (n = 193), with or without ART, exhibit significantly lower levels of cofilin phosphorylation (hyperactivation) than those from healthy controls (n = 100; ratio, 1.1:2.3; P < 0.001); cofilin hyperactivation is also associated with poor CD4 T cell recovery following ART. These results suggest an HIV-mediated systemic dysregulation of T cell motility that cannot be repaired solely by ART. We further demonstrate that stimulating blood CD4 T cells with an anti-human Î±4Î²7 integrin antibody can trigger signal transduction and modulate the cofilin pathway, partially restoring T cell motility in vitro. However, we also observed that severe T cell motility defect caused by high degrees of cofilin hyperactivation was not repairable by the anti-integrin antibody, demonstrating a mechanistic hindrance to restore immune functions in vivo. Our study suggests that cofilin is a key molecule that may need to be therapeutically targeted early for T cell tissue repopulation, immune reconstitution, and immune control of viremia.
Virus and CTL dynamics in the extrafollicular and follicular tissue compartments in SIV-infected macaques
Data from SIV-infected macaques indicate that virus-specific cytotoxic T lymphocytes (CTL) are mostly present in the extrafollicular (EF) compartment of the lymphoid tissue, with reduced homing to the follicular (F) site. This contributes to the majority of the virus being present in the follicle and represents a barrier to virus control. Using mathematical models, we investigate these dynamics. Two models are analyzed. The first assumes that CTL can only become stimulated and expand in the extrafollicular compartment, with migration accounting for the presence of CTL in the follicle. In the second model, follicular CTL can also undergo antigen-induced expansion. Consistent with experimental data, both models predict increased virus compartmentalization in the presence of stronger CTL responses and lower virus loads, and a more pronounced rise of extrafollicular compared to follicular virus during CD8 cell depletion experiments. The models, however, differ in other aspects. The follicular expansion model results in dynamics that promote the clearance of productive infection in the extrafollicular site, with any productively infected cells found being the result of immigration from the follicle. This is not observed in the model without follicular CTL expansion. The models further predict different consequences of introducing engineered, follicular-homing CTL, which has been proposed as a therapeutic means to improve virus control. Without follicular CTL expansion, this is predicted to result in a reduction of virus load in both compartments. The follicular CTL expansion model, however, makes the counter-intuitive prediction that addition of F-homing CTL not only results in a reduction of follicular virus load, but also in an increase in extrafollicular virus replication. These predictions remain to be experimentally tested, which will be relevant for distinguishing between models and for understanding how therapeutic introduction of F-homing CTL might impact the overall dynamics of the infection.
Follicular regulatory T cells are highly permissive to R5-tropic HIV-1
Follicular regulatory T cells (TFR) are a subset of CD4+ T cells in secondary lymphoid follicles. TFR were previously included in the follicular helper T cell (TFH) subset, which are highly permissive to HIV-1. The permissivity of TFR to HIV-1 is unknown. We find TFR are more permissive than TFH to R5-tropic HIV-1 ex vivo TFR expressed more CCR5 and CD4, and supported higher frequencies of viral fusion. Differences in Ki67 expression correlated with HIV-1 replication. Inhibiting cellular proliferation reduced Ki67 expression and HIV-1 replication. Lymph node cells from untreated HIV-infected individuals revealed that TFR harbored the highest concentrations of HIV-1 RNA and highest levels of Ki67 expression. These data demonstrate that TFR are highly permissive to R5-tropic HIV-1 both ex vivo and in vivo This is likely related to elevated CCR5 levels combined with a heightened proliferative state and suggests TFR contribute to persistent R5-tropic HIV-1 replication in vivoImportance In chronic, untreated HIV-1 infection, viral replication is concentrated in secondary lymphoid follicles. Within secondary lymphoid follicles, follicular helper T cells (TFH) have previously been shown to be highly permissive to HIV-1. Recently, another subset of T cells in secondary lymphoid follicles was described, follicular regulatory T cells (TFR). These cells share some phenotypic characteristics with TFH and studies that showed that TFH are highly permissive to HIV-1 included TFR in their definition of TFH. The permissivity of TFR to HIV-1 has not previously been described. Here, we show that TFR are highly permissive to HIV-1 both ex vivo and in vivo The expression of Ki67, a marker of proliferative capacity, is predictive of expression of viral proteins and downregulating Ki67 leads to concurrent decreases in expression of viral proteins. Our study provides new insight into HIV-1 replication and a potential new cell type to target for future treatment.
Potent inhibition of HIV-1 replication in resting CD4 T cells by resveratrol and pterostilbene
HIV-1 infection of resting CD4 T cells plays a crucial and numerically dominant role during virus transmission at mucosal sites and during subsequent acute replication and T cell depletion. Resveratrol and pterostilbene are plant stilbenoids associated with several health promoting benefits. Resveratrol has been shown to inhibit replication of several viruses, including herpes simplex 1 and 2, papillomaviruses, SARS virus and influenza virus. Alone, resveratrol does not inhibit HIV-1 infection of activated T cells, but it does synergize with nucleoside reverse transcriptase inhibitors in these cells to inhibit reverse transcription. Here, we demonstrate that resveratrol and pterostilbene completely block HIV-1 infection at low micromolar dose in resting CD4 T cells, primarily at the reverse transcription step. The anti-HIV effect was fully reversed by exogenous deoxynucleosides and Vpx, a simian immunodeficiency virus protein that increases dNTP levels. These findings are consistent with the reported ability of resveratrol to inhibit ribonucleotide reductase and to lower dNTP levels in cells. This study supports the potential use of resveratrol, pterostilbene or related compounds as adjuvants in anti-HIV pre-exposure prophylaxis (PrEP) formulations.
Pyroptosis, superinfection, and the maintenance of the latent reservoir in HIV-1 infection
A long-lived reservoir of latently infected T cells prevents antiretroviral therapy from eliminating HIV-1 infection. Furthering our understanding of the dynamics of latency generation and maintenance is therefore vital to improve treatment outcome. Using mathematical models and experiments, we suggest that the death of latently infected cells brought about by pyroptosis, or to a lesser extent by superinfection, might be key mechanisms to account for the size and composition of the latent reservoir. Pyroptosis is a form of cell death that occurs in a resting (and thus latently infected) T cell when a productively infected cell attempts cell-to-cell transmission of virus. Superinfection of latently infected cells by productive virus could similarly remove those cells through active virus replication and resulting cytopathicity. The mathematical models presented can explain a number of previously published clinical observations including latent reservoir size and the relationships to viral load in acute HIV infection, measurements of the latent reservoir in chronic infection, and the replacement of wild-type virus by CTL escape mutants within the latent reservoir. Basic virus dynamics models of latency that do not take into account pyroptosis, superinfection, or other potential complexities cannot account for the data.