Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance.

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.

Relative resistance of HIV-1 founder viruses to control by interferon-alpha.

BACKGROUND: Following mucosal human immunodeficiency virus type 1 (HIV-1) transmission, type 1 interferons (IFNs) are rapidly induced at sites of initial virus replication in the mucosa and draining lymph nodes. However, the role played by IFN-stimulated antiviral activity in restricting HIV-1 replication during the initial stages of infection is not clear. We hypothesized that if type 1 IFNs exert selective pressure on HIV-1 replication in the earliest stages of infection, the founder viruses that succeed in establishing systemic infection would be more IFN-resistant than viruses replicating during chronic infection, when type 1 IFNs are produced at much lower levels. To address this hypothesis, the relative resistance of virus isolates derived from HIV-1-infected individuals during acute and chronic infection to control by type 1 IFNs was analysed. RESULTS: The replication of plasma virus isolates generated from subjects acutely infected with HIV-1 and molecularly cloned founder HIV-1 strains could be reduced but not fully suppressed by type 1 IFNs in vitro. The mean IC50 value for IFNα2 (22 U/ml) was lower than that for IFNß (346 U/ml), although at maximally-inhibitory concentrations both IFN subtypes inhibited virus replication to similar extents. Individual virus isolates exhibited differential susceptibility to inhibition by IFNα2 and IFNß, likely reflecting variation in resistance to differentially up-regulated IFN-stimulated genes. Virus isolates from subjects acutely infected with HIV-1 were significantly more resistant to in vitro control by IFNα than virus isolates generated from the same individuals during chronic, asymptomatic infection. Viral IFN resistance declined rapidly after the acute phase of infection: in five subjects, viruses derived from six-month consensus molecular clones were significantly more sensitive to the antiviral effects of IFNs than the corresponding founder viruses. CONCLUSIONS: The establishment of systemic HIV-1 infection by relatively IFNα-resistant founder viruses lends strong support to the hypothesis that IFNα plays an important role in the control of HIV-1 replication during the earliest stages of infection, prior to systemic viral spread. These findings suggest that it may be possible to harness the antiviral activity of type 1 IFNs in prophylactic and potentially also therapeutic strategies to combat HIV-1 infection.

Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling.

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a ß-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1-4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.

Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4+ T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNß that reduced viral replication in vitro by 50% (IC50) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNß resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.

The platelet receptor CLEC-2 is active as a dimer.

The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

Sexually transmitted founder HIV-1 viruses are relatively resistant to Langerhans cell-mediated restriction.

A single HIV-1 variant establishes infection of the host after sexual contact. Identifying the phenotypic characteristics of these Transmitted Founder (T/F) viruses is important to understand the restriction mechanisms during transmission. Langerhans cells (LCs) are the mucosal dendritic cell subset that has been shown to have a protective role in HIV-1 transmission. Immature LCs efficiently capture and degrade HIV-1 via langerin-mediated restriction. Here we have investigated the capacity of T/F HIV-1 strains to infect mucosal Langerhans cells (LCs). Notably, most T/F variants efficiently infected immature LCs derived from skin and vaginal tissue in contrast to chronic HIV-1 laboratory strains. Next we screened a panel of T/F viruses and their matched 6-month consensus sequence viruses. Interestingly most T/F variants infected immature LCs whereas donor-matched 6-month consensus sequence viruses had lost the ability to infect LCs. However, we also identified 6-month consensus sequence viruses that had retained an ability to infect LCs similar to that of the donor-matched T/F virus. Moreover, some T/F viruses and 6-month consensus sequence viruses were unable to infect immature LCs. Further analyses indicated that T/F viruses are less sensitive to langerin-mediated restriction. These data suggest that T/F HIV-1 variants have the ability to infect immature LCs, which will facilitate transmission.