Gaurav D. Gaiha

IFITM3 Inhibits Influenza A Virus Infection by Preventing Cytosolic Entry

To replicate, viruses must gain access to the host cells resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3s actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune systems neutralization of a diverse array of threats.

Association of TRIM22 with the Type 1 Interferon Response and Viral Control during Primary HIV-1 Infection

ABSTRACT Type 1 interferons (IFNs) induce the expression of the tripartite interaction motif (TRIM) family of E3 ligases, but the contribution of these antiviral factors to HIV pathogenesis is not completely understood. We hypothesized that the increased expression of select type 1 IFN and TRIM isoforms is associated with a significantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection. We measured IFN-α, IFN-β, myxovirus resistance protein A (MxA), human TRIM5α (huTRIM5α), and TRIM22 mRNA levels in peripheral blood mononuclear cells (PBMCs) of high-risk, HIV-1-uninfected participants and HIV-1-positive study participants. Samples were available for 32 uninfected subjects and 28 infected persons, all within 1 year of infection. HIV-1-positive participants had higher levels of IFN-β (P = 0.0005), MxA (P = 0.007), and TRIM22 (P = 0.01) and lower levels of huTRIM5α (P < 0.001) than did HIV-1-negative participants. TRIM22 but not huTRIM5α correlated positively with type 1 IFN (IFN-α, IFN-β, and MxA) (all P < 0.0001). In a multivariate model, increased MxA expression showed a significant positive association with viral load (P = 0.0418). Furthermore, TRIM22 but not huTRIM5α, IFN-α, IFN-β, or MxA showed a negative correlation with plasma viral load (P = 0.0307) and a positive correlation with CD4+ T-cell counts (P = 0.0281). In vitro studies revealed that HIV infection induced TRIM22 expression in PBMCs obtained from HIV-negative donors. Stable TRIM22 knockdown resulted in increased HIV-1 particle release and replication in Jurkat reporter cells. Collectively, these data suggest concordance between type 1 IFN and TRIM22 but not huTRIM5α expression in PBMCs and that TRIM22 likely acts as an antiviral effector in vivo.

Comprehensive Identification of Host Modulators of HIV-1 Replication using Multiple Orthologous RNAi Reagents

SUMMARY RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene’s phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1–host cell interactions.

Surfactant Protein A Binds to HIV and Inhibits Direct Infection of CD4+ Cells, but Enhances Dendritic Cell-Mediated Viral Transfer

The identification of surfactant protein A (SP-A) as an important innate immune factor of the lungs, amniotic fluid, and the vaginal tract suggests that it could play an important role during various stages of HIV disease progression and transmission. Therefore, we examined whether SP-A could bind to HIV and also had any effect on viral infectivity. Our data demonstrate that SP-A binds to HIV in a calcium-dependent manner that is inhibitable by mannose and EDTA. Affinity capture of the HIV viral lysate reveals that SP-A targets the envelope glycoprotein of HIV (gp120), which was confirmed by ELISA using recombinant gp120. Digestion of gp120 with endoglycosidase H abrogates the binding of SP-A, indicating that the high mannose structures on gp120 are the target of the collectin. Infectivity studies reveal that SP-A inhibits the infection of CD4+ T cells by two strains of HIV (BaL, IIIB) by >80%. Competition assays with CD4 and mAbs F105 and b12 suggest that SP-A inhibits infectivity by occlusion of the CD4-binding site. Studies with dendritic cells (DCs) demonstrate that SP-A enhances the binding of gp120 to DCs, the uptake of viral particles, and the transfer of virus from DCs to CD4+ T cells by >5-fold at a pH representative of the vaginal tract. Collectively, these results suggest that SP-A acts as a dual modulator of HIV infection by protecting CD4+ T cells from direct infection but enhancing the transfer of infection to CD4+ T cells mediated by DCs.

Dysfunctional HIV-specific CD8+ T cell proliferation is associated with increased caspase-8 activity and mediated by necroptosis

Decreased HIV-specific CD8(+) T cell proliferation is a hallmark of chronic infection, but the mechanisms of decline are unclear. We analyzed gene expression profiles from antigen-stimulated HIV-specific CD8(+) T cells from patients with controlled and uncontrolled infection and identified caspase-8 as a correlate of dysfunctional CD8(+) T cell proliferation. Caspase-8 activity was upregulated in HIV-specific CD8(+) T cells from progressors and correlated positively with disease progression and programmed cell death-1 (PD-1) expression, but negatively with proliferation. In addition, progressor cells displayed a decreased ability to upregulate membrane-associated caspase-8 activity and increased necrotic cell death following antigenic stimulation, implicating the programmed cell death pathway necroptosis. In vitro necroptosis blockade rescued HIV-specific CD8(+) T cell proliferation in progressors, as did silencing of necroptosis mediator RIPK3. Thus, chronic stimulation leading to upregulated caspase-8 activity contributes to dysfunctional HIV-specific CD8(+) T cell proliferation through activation of necroptosis and increased cell death.

Surfactant protein D modulates HIV infection of both T-cells and dendritic cells

Surfactant Protein D (SP-D) is an oligomerized C-type lectin molecule with immunomodulatory properties and involvement in lung surfactant homeostasis in the respiratory tract. SP-D binds to the enveloped viruses, influenza A virus and respiratory syncytial virus and inhibits their replication in vitro and in vivo. SP-D has been shown to bind to HIV via the HIV envelope protein gp120 and inhibit infectivity in vitro. Here we show that SP-D binds to different strains of HIV (BaL and IIIB) and the binding occurs at both pH 7.4 and 5.0 resembling physiological relevant pH values found in the body and the female urogenital tract, respectively. The binding of SP-D to HIV particles and gp120 was inhibited by the presence of several hexoses with mannose found to be the strongest inhibitor. Competition studies showed that soluble CD4 and CVN did not interfere with the interaction between SP-D and gp120. However, soluble recombinant DC-SIGN was shown to inhibit the binding between SP-D and gp120. SP-D agglutinated HIV and gp120 in a calcium dependent manner. SP-D inhibited the infectivity of HIV strains at both pH values of 7.4 and 5.0 in a concentration dependent manner. The inhibition of the infectivity was abolished by the presence of mannose. SP-D enhanced the binding of HIV to immature monocyte derived dendritic cells (iMDDCs) and was also found to enhance HIV capture and transfer to the T-cell like line PM1. These results suggest that SP-D can bind to and inhibit direct infection of T-cells by HIV but also enhance the transfer of infectious HIV particles from DCs to T-cells in vivo.

The Fiery Side of HIV–Induced T Cell Death

A cytosolic protein that senses fragments of HIV-1 DNA triggers the death of uninfected CD4 T cells. [Also see Report by Monroe et al.] In untreated HIV-1 infection, the progressive destruction of CD4 T cells gives rise to a devastating number of opportunistic infections—the hallmark of AIDS. Yet, it remains unclear how HIV-1 inexorably depletes these essential immune cells. Most of the dying CD4 T cells are uninfected “bystanders” that self-destruct upon exposure to HIV-1 DNA products generated during aborted infection (1). Doitsh et al. (2) and a report by Monroe et al. on page 428 in this issue (3) show that these truncated fragments of HIV-1 DNA trigger CD4 T cell demise through an intense inflammatory cell death pathway—called pyroptosis—after their recognition by an intracellular DNA sensor (4, 5).

Identification of CD8+ T Cell Host Factors Involved in HIV Control

Methods To accomplish this, we performed whole genome transcriptional profiling on sorted epitope-specific CD8 T cells, in the presence and absence of cognate peptide, from a total of 8 B*2705 elite controllers and progressors. Epitope-specific CTLs were isolated using HLA class I tetramers and used to prepare mRNA for evaluation by gene expression profiles using whole genome Illumina bead arrays. Bulk CD8 T cells were used as an internal control for each patient.