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

SummaryRNAi 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 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

IFITM3 Inhibits Influenza A Virus Infection by Preventing Cytosolic Entry

To replicate, viruses must gain access to the host cell's 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 IFITM3's 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 system's neutralization of a diverse array of threats

Studies on the interactions between surfactant proteins A and D and HIV

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Studies on the interactions between surfactant proteins A and D and HIV

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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 DC

Surfactant protein A binds to HIV and inhibits direct infection of CD4[sup]+ 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

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

SP-D enhances binding of gp120 to iMDDC and enhances HIV capture and transfer by iMDDCs.

<p>Binding of FITC-labeled gp120 to iMDDCs in the presence and absence of SP-D (5 µg/mL) at <b>A</b>: pH of 7.4 or 5.0. DCs were incubated with FITC-labeled gp120, or FITC-gp120 and SP-D at 4 °C for 1 h and then washed extensively before analysis by flow cytometry. The iMDDCs were initially analysed in FACS dot plot showing size (FSC-H) and granularity (SSC-H) characteristics. A gate (R1) was used to select for immature DCs and these were further analysed for binding of the FITC labeled gp120 or FITC labeled gp120 in the presence of SP-D. A geometric mean fluorescence intensity (GMFI) was calculated for each histogram plot and statistical analysis was performed using an unpaired t-test with Welch's correction. * shows statistically significant increase in p24 antigen uptake by DCs in the presence of SP-D compared to the uptake with no SP-D present (p <0.05). N.s indicates no statistically significance between gp120 and the presence of SP-D and no SP-D present (p = 0.409). <b>B</b>: Virus captured by iMDDCs in the presence or absence of SP-D (5 µg/mL). DCs were incubated with AT-2 inactivated HIV BaL particles for 2 h at 37 °C, washed extensively in 10 mM EDTA containing buffer, and lysed for analysis by p24 ELISA. The value of virus captured by iMDDCs in the absence of SP-D at pH 7.4 was defined as 100%. Each bar represents the mean ± S.D. (n = 3). *shows statistically significant increase in p24 antigen uptake by DCs in the presence of SP-D compared to no SP-D present (p <0.05). <b>C</b>: Infectious HIV BaL was incubated with iMDDCs in the presence or absence of the indicated concentrations of SP-D (0–10 µg/mL) at a pH of 7.4 or 5.0. Unbound virus was removed by washing and iMDDCs were then co-cultured with PM1 cells for five days before levels of p24 antigen in the culture supernatants was measured by ELISA. The value of virus transferred by iMDDCs to PMI cells in the absence of SP-D at pH 7.4 was defined as 100%. *shows statistically significant increase in p24 antigen uptake by DCs in the presence of SP-D compared to no SP-D present (p <0.05).</p