Cell-free HIV-1 Infection Research Articles

Antithetic effect of interferon-α on cell-free and cell-to-cell HIV-1 infection.

In HIV-1-infected individuals, transmitted/founder (TF) virus contributes to establish new infection and expands during the acute phase of infection, while chronic control (CC) virus emerges during the chronic phase of infection. TF viruses are more resistant to interferon-alpha (IFN-α)-mediated antiviral effects than CC virus, however, its virological relevance in infected individuals remains unclear. Here we perform an experimental-mathematical investigation and reveal that IFN-α strongly inhibits cell-to-cell infection by CC virus but only weakly affects that by TF virus. Surprisingly, IFN-α enhances cell-free infection of HIV-1, particularly that of CC virus, in a virus-cell density-dependent manner. We further demonstrate that LY6E, an IFN-stimulated gene, can contribute to the density-dependent enhancement of cell-free HIV-1 infection. Altogether, our findings suggest that the major difference between TF and CC viruses can be explained by their resistance to IFN-α-mediated inhibition of cell-to-cell infection and their sensitivity to IFN-α-mediated enhancement of cell-free infection.

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

ABSTRACT Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell–cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

Environmental Restrictions: A New Concept Governing HIV-1 Spread Emerging from Integrated Experimental-Computational Analysis of Tissue-Like 3D Cultures.

HIV-1 can use cell-free and cell-associated transmission modes to infect new target cells, but how the virus spreads in the infected host remains to be determined. We recently established 3D collagen cultures to study HIV-1 spread in tissue-like environments and applied iterative cycles of experimentation and computation to develop a first in silico model to describe the dynamics of HIV-1 spread in complex tissue. These analyses (i) revealed that 3D collagen environments restrict cell-free HIV-1 infection but promote cell-associated virus transmission and (ii) defined that cell densities in tissue dictate the efficacy of these transmission modes for virus spread. In this review, we discuss, in the context of the current literature, the implications of this study for our understanding of HIV-1 spread in vivo, which aspects of in vivo physiology this integrated experimental–computational analysis takes into account, and how it can be further improved experimentally and in silico.

Number of infection events per cell during HIV-1 cell-free infection

HIV-1 accumulates changes in its genome through both recombination and mutation during the course of infection. For recombination to occur, a single cell must be infected by two HIV strains. These coinfection events were experimentally demonstrated to occur more frequently than would be expected for independent infection events and do not follow a random distribution. Previous mathematical modeling approaches demonstrated that differences in target cell susceptibility can explain the non-randomness, both in the context of direct cell-to-cell transmission, and in the context of free virus transmission (Q. Dang et al., Proc. Natl. Acad. Sci. USA 101:632-7, 2004: K. M. Law et al., Cell reports 15:2711-83, 2016). Here, we build on these notions and provide a more detailed and extensive quantitative framework. We developed a novel mathematical model explicitly considering the heterogeneity of target cells and analysed datasets of cell-free HIV-1 single and double infection experiments in cell culture. Particularly, in contrast to the previous studies, we took into account the different susceptibility of the target cells as a continuous distribution. Interestingly, we showed that the number of infection events per cell during cell-free HIV-1 infection follows a negative-binomial distribution, and our model reproduces these datasets.

Cell-Free versus Cell-to-Cell Infection by Human Immunodeficiency Virus Type 1 and Human T-Lymphotropic Virus Type 1: Exploring the Link among Viral Source, Viral Trafficking, and Viral Replication.

Human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1) are complex retroviruses mainly infecting CD4(+) T lymphocytes. In addition, antigen-presenting cells such as dendritic cells (DCs) are targeted in vivo by both viruses, although to a lesser extent. Interaction of HIV-1 with DCs plays a key role in viral dissemination from the mucosa to CD4(+) T lymphocytes present in lymphoid organs. While similar mechanisms may occur for HTLV-1 as well, most HTLV-1 data were obtained from T-cell studies, and little is known regarding the trafficking of this virus in DCs. We first compared the efficiency of cell-free versus cell-associated viral sources of both retroviruses at infecting DCs. We showed that both HIV-1 and HTLV-1 cell-free particles are poorly efficient at productively infecting DCs, except when DC-SIGN has been engaged. Furthermore, while SAMHD-1 accounts for restriction of cell-free HIV-1 infection, it is not involved in HTLV-1 restriction. In addition, cell-free viruses lead mainly to a nonproductive DC infection, leading to trans-infection of T-cells, a process important for HIV-1 spread but not for that of HTLV-1. Finally, we show that T-DC cell-to-cell transfer implies viral trafficking in vesicles that may both increase productive infection of DCs ("cis-infection") and allow viral escape from immune surveillance. Altogether, these observations allowed us to draw a model of HTLV-1 and HIV-1 trafficking in DCs.

Macrophage infection via selective capture of HIV-1-infected CD4+ T cells.

SummaryMacrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4+ T cells leading to efficient macrophage infection. Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.

Breast Milk from Tanzanian Women Has Divergent Effects on Cell-Free and Cell-Associated HIV-1 Infection In Vitro

Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-LewisX from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding.

Milk Matters: Soluble Toll-Like Receptor 2 (sTLR2) in Breast Milk Significantly Inhibits HIV-1 Infection and Inflammation

The majority of infants who breastfeed from their HIV-positive mothers remain uninfected despite constant and repeated exposure to virus over weeks to years. This phenomenon is not fully understood but has been closely linked to innate factors in breast milk (BM). Most recently we have focused on one such innate factor, soluble Toll-like receptor 2 (sTLR2) for its significant contribution as an inhibitor of inflammation triggered by bacterial and viral antigens. We hypothesized that sTLR2 in BM inhibits immune activation/inflammation and HIV-1 infection. sTLR2 protein profiles were analyzed in HIV-uninfected BM and showed dramatic variability in expression concentration and predominant sTLR2 forms between women. sTLR2 immunodepleted BM, versus mock-depleted BM, incubated with Pam3CSK4 lead to significant increases in IL-8 production in a TLR2-dependant fashion in U937, HEK293-TLR2, and Caco-2. Importantly, TLR2-specific polyclonal and monoclonal antibody addition to BM prior to cell-free R5 HIV-1 addition led to significantly (P<0.01, P<0.001, respectively) increased HIV-1 infection in TZM-bl reporter cells. To confirm these findings, sTLR2-depletion in BM led to significantly (P<0.001) increased HIV-1 infection in TZM-bl cells. Notably, immunodepletion does not allow for the complete removal of sTLR2 from BM, thus functional testing shown here may underestimate the total effect elicited by sTLR2 against HIV-1 and synthetic bacterial ligand. This study provides evidence for the first time that sTLR2 in BM may provide a dual protective role for infants breastfeeding from their HIV-infected mothers by; (1) immunomodulating pro-inflammatory responses to bacterial ligands, and (2) directly inhibiting cell-free HIV-1 infection. Thus, sTLR2 in BM may be critical to infant health and prove beneficial in decreasing vertical HIV-1 transmission to infants.

Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

BackgroundThe human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route.ResultsHere we analysed viral entry in wild type BeWo (CCR5+, CXCR4+) and BeWo-CD4+ (CD4+, CCR5+, CXCR4+) cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted.ConclusionCollectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

Distinct efficacy of HIV-1 entry inhibitors to prevent cell-to-cell transfer of R5 and X4 viruses across a human placental trophoblast barrier in a reconstitution model in vitro

Background and methodsHIV-1 cell-to-cell transmission is more efficient than infection of permissive cells with cell-free particles. The potency of HIV-1 entry inhibitors to inhibit such transmission is not well known. Herein, we evaluated the efficacy of this new class of antiretrovirals to block cell-to-cell transmission of HIV-1 in a model of reconstitution of the human placental trophoblast barrier in vitro.ResultsOur data show that CCR5 antagonists and T20 inhibit the passage of the virus across the BeWo cell monolayer in contact with PBMCs infected with an R5 (Ba-L) and a dualtropic (A204) HIV-1 with IC50s in the range of 100 – 5,000 nM for TAK779; 90 to 15,000 nM for SCH-350581 and 3,000 to 20,000 nM for T20. The CXCR4 antagonist AMD3100 is also effective against X4 HIV-1 infected PBMCs in our model with IC50 comprised between 4 nM and 640 nM. HIV-1 entry inhibitors are less efficient to block cell-to-cell virus transmission than cell-free HIV-1 infection of PBMCs and CCR5 antagonists do not prevent PBMC infection by dual tropic HIV-1 in contrast to cell-to-cell infection in our model.Surprisingly, T20 (and C34) do not block cell-to-cell transmission of X4 HIV-1 but, rather, increase 80 to 140 fold, compared to control without drug, the passage of the virus across the trophoblast barrier. Additional experiments suggest that the effect of T20 on BeWo/PBMC-X4 HIV-1 is due to an increase of effector-target cells fusion.ConclusionOur results support further evaluation of HIV-1 coreceptor antagonists, alone or combined to other antiretrovirals, in a perspective of prevention but warn on the use of T20 in patients bearing X4 HIV-1 at risk of transmission.

Cell-Free HIV Type 1 Infection is Restricted in the Human Trophoblast Choriocarcinoma BeWo Cell Line, Even with Expression of CD4, CXCR4 and CCR5

The restriction of cell-free HIV-1 infection has been demonstrated in placental trophoblast choriocarcinoma BeWo cells. We tried to determine the level of the viral replication cycle at which this restriction occurs. BeWo cells produce infectious viruses after transfection with HIV-1 plasmids, independently of viral tropism. CCR5 and CXCR4, but not the CD4 molecule, were detected at the cell surface. We therefore derived CD4-expressing clones from transfected BeWo cells. Cell-free virus infection of these clones resulted in neither virus production nor viral sequence integration, indicating that the restriction occurs before integration of the virus. If we used luciferase reporter viruses pseudotyped with HIV-1 Env R5 and X4 for infection, no luciferase activity was detected, even in the BeWo-CD4+ clone, in contrast to what was observed in VSV-G pseudotyped virus infection. Our results show that infection of trophoblast-derived cells with cell-free virus is at least restricted at the level of entry. Thus, BeWo is an interesting human placental cell line that is resistant to HIV-1, even if CD4, CXCR4, and CCR5 are expressed.

MAP 30: a new inhibitor of HIV-1 infection and replication

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti-HIV Protein), is a basic protein of about 30 kDa. It exhibits dose-dependent inhibition of cell-free HIV-1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM-ss monolayers; (II) viral core protein p24 expression; and (iii) viral-associated reverse transcriptase (RT) activity in HIV-1 infected H9 cells. The doses required for 50% inhibition (ID 50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV-1 infections. The sequence of the N-terminal 44 amino acids of MAP 30 has been determined.

CD11a/CD 18 (LFA-1) Epitopes Involved in Syncytium Formation Among CD4+T-Cells Following Cell Free HIV-1 Infection

Like particular monoclonal antibodies to CD4, monoclonal antibodies to epitopes localized at the top of the alpha-subunit (CD11a) and at the stem of the beta-subunit (CD18) of the lymphocyte function associated antigen-1 (LFA-1) block syncytium formation when added to CD4+ T-cells before or shortly after (5 min) cell free HIV-1 is added. This indicates involvement of LFA-1 epitopes in the early stage of cell-free infection. Syncytium formation is still blocked by CD4 antibodies when added two hours after virus adsorption, CD11a and CD18 antibodies are ineffective at this late stage. Radio-immune precipitation experiments in acutely infected T-cells with the appropriate CD11a and CD18 antibodies suggested a link between CD18 and CD4 in the cell lines studied, possibly mediated by the CD3/TCR complex. In chronically infected cells, where little CD4 is expressed, LFA-1 antibodies still precipitate the viral envelope, pointing to a direct LFA-1 envelope interaction in that late stage of infection. Our results indicate that a limited number of LFA-1 epitopes is involved in syncytium formation among T-cells, none of which is required for virus entry in the cell or virus spread in the culture.