Identification of potential HIV restriction factors by combining evolutionary signatures with functional analyses

Restrictions factors are host cell encoded proteins that interfere with multiple steps of human immunodeficiency virus (HIV) replication to inhibit viral spread. These antiretroviral factors are commonly under positive selection, upregulated during HIV-1 infection, and/or frequently interacting with viral proteins. Here, I combined evolutionary genomics, transcriptomics, and protein interaction data to identify thirty cellular genes sharing these characteristic features of known restriction factors. Cotransfection of a high proportion of candidate genes with HIV-1 proviruses impaired the production of infectious HIV-1 without affecting cell viability. Among them were members of the APOL and TNFR superfamilies, as well as CD164 that suppressed HIV-1 production by more than 90%. In the absence of the accessory viral proteins Vpr, Vpu and Nef, which are known to antagonize antiviral factors, additional candidate factors such as GBP5, IFI16, and SPN achieved more than 90% reduction. Notably, this screening approach also led to the identification of two genes (IL-1A and SP110) that enhanced HIV-1 production. Titration experiments for several factors confirmed their impact on HIV-1 and flow cytometric analyses, p24 capsid ELISA and Western blot revealed that many candidate genes influenced HIV-1 gene transcription and/or translation. Many factors exerted inhibitory effects on viral promoters not limited to the HIV-1 promoter. In addition, Western blot analyses demonstrated that CD164, CD1A, CD3G, GBP5, OAS1 and SPN might reduce virion infectivity by interfering with Env function. In summary, my results show that the number of human genes sharing the characteristics of known HIV restriction factors is very limited and are a first step towards clarifying how many of them display specific antiretroviral activity. Further analyses will reveal which of the newly identified candidate restriction factors inhibit HIV-1 replication in primary human cells

Influenza A virus does not encode a tetherin antagonist with Vpu-like activity and induces IFN-dependent tetherin expression in infected cells.

The interferon-induced host cell factor tetherin inhibits release of human immunodeficiency virus (HIV) from the plasma membrane of infected cells and is counteracted by the HIV-1 protein Vpu. Influenza A virus (FLUAV) also buds from the plasma membrane and is not inhibited by tetherin. Here, we investigated if FLUAV encodes a functional equivalent of Vpu for tetherin antagonism. We found that expression of the FLUAV protein NS1, which antagonizes the interferon (IFN) response, did not block the tetherin-mediated restriction of HIV release, which was rescued by Vpu. Similarly, tetherin-mediated inhibition of HIV release was not rescued by FLUAV infection. In contrast, FLUAV infection induced tetherin expression on target cells in an IFN-dependent manner. These results suggest that FLUAV escapes the antiviral effects of tetherin without encoding a tetherin antagonist with Vpu-like activity

NF-κB inhibition by SIVolc and SIVcol Vpr reduces LTR-driven gene expression.

<p>(A, B) HEK293T cells were cotransfected with the indicated <i>vpr</i> alleles, a firefly luciferase reporter construct under the control of the HIV-1 M, SIVcol or SIVolc LTR promoter, and a <i>Gaussia</i> luciferase construct for normalization. Cells were (A) stimulated with TNFα or (B) cotransfected with a constitutively active mutant of IKKβ (c.a. IKKβ). Luciferase activities were determined 40 hr post-transfection. Mean values of four independent experiments in triplicates ± SEM are shown (*p<0.05; **p < 0.01; ***p < 0.001).</p

Inhibition of IFNβ promoter activity by SIVcol and SIVolc Vpr.

<p>(A) Schematic representation of the canonical NF-κB signaling pathway and the IFNβ promoter reporter constructs used in the present study. The wild type IFNβ core promoter contains an NF-κB binding site that was mutated by introducing three nucleotide changes (highlighted in red). (B) HEK293T cells were cotransfected with different <i>vpr</i> alleles, a <i>Gaussia</i> luciferase construct for normalization, and one of the firefly luciferase reporter constructs described in (A) (with wild type or mutated NF-κB binding site). To activate the IFNβ promoter, cells were stimulated with Sendai virus. Luciferase activities were determined 40 hr post-transfection. Vprs were categorized into three groups: Vprs from lentiviruses encoding <i>vpu</i> (HIV/SIV_<i>vpu</i>, green), downmodulating CD3 via Nef (HIV/SIV_CD3, blue), or lacking a <i>vpu</i> gene and the CD3-downmodulation activity (SIVolc/col, orange) (**p < 0.01; ***p < 0.001; n.s. p>0.05).</p

Virion-associated Vpr does not affect the activation of NF-κB in infected T cells.

<p>(A-D) SupD1 cells were transduced with the indicated VSV-G pseudotyped CH293.1 constructs. (A) HIV-1 CH293, (B) SIVwrc, (C) SIVolc, or (D) SIVgsn Vpr were either delivered <i>in cis</i> (<i>i</i>.<i>e</i>. incorporated in viral particles AND encoded in the viral genome) or <i>in trans</i> (<i>i</i>.<i>e</i>. incorporated in viral particles, but not encoded in the viral genome). CH293.1 wild type and CH293.1 <i>vpu</i>- <i>vpr</i>- served as controls. Cells were harvested at the indicated time points post-transduction to determine the activation levels of NF-κB. The mean values of triplicate infections ± SD are shown. Asterisks indicate a statistically significant difference compared to the mock control (*p<0.05; **p < 0.01; ***p < 0.001).</p

Vpu-, Vpr- and Nef-mediated inhibition of immune activation.

<p>The left panel illustrates the emergence of Vpu-, Vpr- and Nef-mediated suppression of immune activation during primate lentiviral evolution: Most primate lentiviruses use their Nef proteins to prevent T cell activation via downmodulation of CD3 (blue). HIV-1, SIVcpz/gor as well as SIVgsn/mon/mus encode a <i>vpu</i> gene and evolved Vpu-mediated inhibition of NF-κB activation (green). Nef-mediated CD3 downmodulation was lost in these viruses (violet star). In SIVcol and SIVolc, the loss of CD3 downmodulation by Nef is associated with the evolution of Vpr-mediated suppression of NF-κB signaling (orange). The tree is a schematic representation of primate lentiviral evolution and based on previous phylogenetic analyses [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006598#ppat.1006598.ref012" target="_blank">12</a>]; branch lengths do not correlate with phylogenetic relationships. While Nef directly targets the CD3 receptor to prevent downstream NF-κB signaling, Vpu and SIVcol/SIVolc Vpr target the canonical NF-κB signaling cascade further downstream, independently of the receptor (right panels). Some Vpu proteins additionally counteract the host restriction factor Tetherin, which also acts as an immune sensor.</p

SIVcol and SIVolc Nef fail to efficiently downmodulate the T cell receptor CD3.

<p>(A) Human PBMCs were transduced with VSV-G pseudotyped NL4-3 constructs coexpressing the indicated Nef proteins and eGFP via an IRES. 72 hr post-transduction, CD3 surface expression was quantified by flow cytometry. Mean values of six infections ± SEM are shown on the left. The panel on the right shows examples for primary flow cytometry data. (B) Due to the lack of an antibody detecting SIVcol and SIVolc Nef, counteraction of human SERINC5 was analyzed to verify functional Nef expression. HEK293T cells were cotransfected with increasing amounts of a SERINC5 expression plasmid and the NL4-3-based proviral constructs also used in (A). 40 hr post-transfection, infectious virus yield was quantified by infection of TZM-bl reporter cells. Mean values of three independent experiments in triplicates ± SEM are shown. (C) HEK293T cells were cotransfected with the proviral constructs described in (A) and expression vectors for CD3-CD8 fusion proteins comprising the cytoplasmic domain of human or colobus CD3ζ and the extracellular and transmembrane domain of human CD8. 40 hr post transfection, CD3ζ downmodulation was determined by monitoring CD8 surface levels via flow cytometry. Mean values of three to four independent experiments ± SEM are shown. In (A) and (C), asterisks indicate statistically significant differences compared to the <i>nef</i>-defective control (**p < 0.01; ***p < 0.001).</p

Vpr-mediated modulation of NF-κB activity and immune activation in infected T cells.

<p>(A) Genomic organization of a chimeric infectious molecular clone (IMC) of HIV-1 M CH293.1 expressing heterologous <i>vpr</i> alleles. The CH293 <i>vpr</i> open reading frame was replaced by XbaI and MluI restriction sites (green), allowing the insertion of heterologous AU1-tagged <i>vpr</i> alleles (yellow). The original <i>vpr</i> start codon in <i>vif</i> was silenced (blue) to prevent expression of a truncated CH293 Vpr. Expression of Vpu was abrogated by inserting a premature stop codon (pink). (B) Expression of heterologous Vpr proteins from the CH293.1 chimeras described in (A). HEK293T cells were cotransfected with CH293.1 IMCs encoding the indicated <i>vpr</i> alleles. 40 hr post-transfection, cells and supernatants were harvested and virions in the supernatant were purified by centrifugation through a sucrose cushion. Subsequently, Western blotting was performed to detect AU1-tagged Vpr, Env and Gag. Detection of GAPDH served as loading control. (C) HEK293T cells were cotransfected with the indicated CH293.1 chimeras, a firefly luciferase reporter construct under the control of three NF-κB binding sites, and a <i>Gaussia</i> luciferase construct for normalization. Luciferase activities were determined 40 hr post-transfection. Mean values of three independent experiments in triplicates ± SEM are shown. (D) The SupD1 NF-κB reporter cell line was transduced with the indicated VSV-G pseudotyped CH293.1 chimeras. Cells were harvested at various time points post-transduction to determine the activation levels of NF-κB. The mean values of triplicate infections ± SD of a representative experiment are shown. (E) PBMCs of three different donors were transduced with VSV-G pseudotyped CH293.1 chimeras encoding the indicated <i>vpr</i> alleles. Cells were harvested 72 hr post-transduction and total cellular RNA was isolated and reversely transcribed. IFNβ mRNA levels were determined by quantitative RT-PCR and normalized to GAPDH mRNA. The mean values ± SEM are shown. In (C) to (E), asterisks indicate statistically significant differences compared to CH293.1 wild type transfected or infected cells (*p < 0.05; **p < 0.01; ***p < 0.001).</p

Modulation of NF-κB activity by primate lentiviral Vpr proteins.

<p>HEK293T cells were cotransfected with the indicated <i>vpr</i> alleles, a firefly luciferase reporter construct under the control of three NF-κB binding sites, and a <i>Gaussia</i> luciferase construct for normalization. In the middle and bottom panels, cells were additionally stimulated with TNFα or cotransfected with a constitutively active mutant of IKKβ (c.a. IKKβ), respectively. Luciferase activities were determined 40 hr post-transfection. All values (including the no inducer controls) are shown as percentage of the vector control, which was set to 100%. Mean values of three to ten independent experiments in triplicates ± SEM are shown. Asterisks indicate statistically significant differences compared to the vector control (*p<0.05; **p < 0.01; ***p < 0.001). In the panels on the right, Vprs were divided into three groups: Vprs from lentiviruses encoding <i>vpu</i> (HIV/SIV_<i>vpu</i>, green), downmodulating CD3 via Nef (HIV/SIV_CD3, blue), or lacking a <i>vpu</i> gene and the CD3-downmodulation activity (SIVolc/col, orange). Whiskers of the boxplots indicate the 5^th and 95^th percentiles.</p