SERINC5 Mediates a Postintegration Block to HIV-1 Gene Expression in Macrophages

Abstract

HIV-1 antagonizes SERINC5 by redundant mechanisms, primarily through Nef and additionally via envelope glycoprotein. Paradoxically, HIV-1 preserves Nef function to ensure the exclusion of SERINC5 from virion incorporation regardless of the availability of envelope that can confer resistance, suggesting additional roles of the virion-incorporated host factor. Here, we report an unusual mode of SERINC5 action in inhibiting viral gene expression. This inhibition is observed only in the myeloid lineage cells but not in the cells of epithelial or lymphoid origin. We found that SERINC5-bearing viruses induce the expression of RPL35 and DRAP1 in macrophages, and these host proteins intercept HIV-1 Tat from binding to and recruiting a mammalian capping enzyme (MCE1) to the HIV-1 transcriptional complex. As a result, uncapped viral transcripts are synthesized, leading to the inhibition of viral protein synthesis and subsequent progeny virion biogenesis. Cell-type-specific inhibition of HIV-1 gene expression thus exemplifies a novel antiviral function of virion-incorporated SERINC5. IMPORTANCE In addition to Nef, HIV-1 envelope glycoprotein has been shown to modulate SERINC5-mediated inhibition. Counterintuitively, Nef from the same isolates preserves the ability to prevent SERINC5 incorporation into virions, implying additional functions of the host protein. We identify that virion-associated SERINC5 can manifest an antiviral mechanism independent of the envelope glycoprotein to regulate HIV-1 gene expression in macrophages. This mechanism is exhibited by affecting the viral RNA capping and is plausibly adopted by the host to overcome the envelope glycoprotein-mediated resistance to SERINC5 restriction.

Keywords: SERINC5; contextualized protein-protein interactions; macrophages; postentry block to viral protein synthesis; protein interactome remodeling; viral RNA capping.

FIG 1

Target cell-type specific infectivity inhibition by SERINC5. (A) Overview of the experimental setup. (B) Indicated cell lines were infected with Nef-defective HIV-1 produced by HEK293T by cotransfecting VSV-G, NL4-3, Luciferase Env(−) R(−), SERINC5, or the equivalent empty vector. Infectivity was normalized to reverse transcriptase (RT) units. The values are means (n = 3) and SD. (C) THP-1-derived macrophages were infected with Nef-defective HIV-1 (zsgreen reporter) as in panel B. Infectivity was normalized to RT units. The values are means (n = 3) and SD. An unpaired t test was used. Lower panels, representative infectivity images from the upper panel. Bar, 200 μm. (D) THP-1-derived macrophages were infected with Nef-defective HIV-1 with increasing amounts of RT units. The values are means (n = 3) and SD. (E) THP-1-derived macrophages and TZM-GFP reporter cells were infected with Nef-defective HIV-1 produced in HEK293T by cotransfecting JR-FL env, NL4-3, Luciferase Env(−) R(−), SERINC5, or the equivalent empty vector. Infectivity was normalized to RT units. The values are means (n = 3) and SD. An unpaired t test was used. (F) THP-1-derived macrophages and TZM-GFP reporter cells were infected with Nef-defective HIV-1 produced by Jurkat TAg and Jurkat TAg ^SER5−/− by cotransfecting VSV-G env and NL4-3, Luciferase Env(−) R(−). Infectivity was normalized to RT units. The values are means (n = 3) and SD. An unpaired t test was used. (G) Quantification of IRF3 activation (Luciferase activity) from the supernatant of THP-1 Dual cells upon challenge with RT-normalized HIV-1 (SERINC5[−] and SERINC5[+]) bearing HXB2, JRFL, or VSV-G envelopes. The values are means (n = 3) and SD. An unpaired t test was used. (H) Primary CD4⁺ T cells and CD14⁺-derived macrophages obtained from three different healthy donors, enriched to purity (Fig. S7), were infected as in panel B. The values are means (n = 3) and SD. An unpaired t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 2

SERINC5 virion association renders the particles defective in THP-1. (A) Infectivity of HIV-1 SERINC5+/− virions in THP-1-derived target macrophages that were primed with either conditioned media (CM) or VSV-G-enveloped vesicles prior to infection with Nef-defective HIV-1. Infectivity was normalized to RT units. Vesicles were produced by transfection of envelope glycoprotein along with empty vector (SERINC5−) or vector expressing SERINC5 (SERINC5+). The values are means (n = 3) and SD. An unpaired t test was used. (B) THP-1-derived macrophages infected with HIV-1 produced from HEK293T by cotransfecting pMD2.G, NL4-3, Luciferase Env(−) R(−), SERINC5, or equivalent empty vector, along with HIV-1 Nef-HA/MLV-HA-GlycoGag/empty vector. Infectivity was normalized to RT units. Lower panel, Western blot showing C-terminally FLAG-tagged SERINC5 and HA-tagged Nef Lai and GlycoGag with the corresponding β-actin from virus-producing cell lysate (HEK293T). The values are means (n = 3) and SD. An unpaired t test was used. (C) THP-1-derived macrophages were infected with HIV-1 produced as in panel B with HIV-1 Lai Nef or indicated Nef mutants. Infectivity was normalized to RT units. Lower panel, Western blot showing C-terminally FLAG-tagged SERINC5 and HA-tagged WT and mutants of Nef Lai with the corresponding β-actin from cell lysate (HEK293T). The values are means (n = 3) and SD. An unpaired t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; HA, hemagglutinin; MLV, murine leukemia virus; ns, not significant.

FIG 3

A postintegration block to HIV-1 infectivity in THP-1 macrophages. (A) Representative figure for nlsCre assay to quantify viral fusion. Lower panel, representative image acquired after the assay on the high-content screening (HCS) platform. The nuclei of THP-1 Lox red fluorescent protein (RFP) cells are counterstained with Hoechst, and the RFP signal is visualized in infected cells. Bar, 100 μm. (B) HIV-1 Cre particles produced from HEK293T by cotransfection of VSV-G, 8.9 Cre, SERINC5, or equivalent empty vector were used for scoring fusion efficiency under indicated conditions. Cre-delivery-based fusion was quantified in THP-1 macrophages expressing a Cre-sensitive RFP reporter by scoring the RFP-positive cells. The counts were normalized to RT units. The target cells were treated with either dimethyl sulfoxide (DMSO), zidovudine (AZT), or bafilomycin. The values are means (n = 3) and SD. An unpaired t test was used. (C) THP-1-derived macrophages were infected with Nef-defective HIV-1 produced from HEK293T by cotransfecting pMD2.G, NL4-3, Luciferase Env(−) R(−), SERINC5, or equivalent empty vector. Early reverse transcription products (early RT) from infected cells (treated with DMSO or AZT) were quantified using quantitative PCR (qPCR) at indicated time points. Lower panel, late reverse transcription products (denoted late RT) from infected THP-1 macrophages (treated with DMSO or AZT) were quantified using qPCR at 24 h postinfection (hpi). The values are means (n = 3) and SD. An unpaired t test was used. (D) THP-1-derived macrophages infected with Nef-defective HIV-1 produced as in panel B were treated with DMSO or AZT and used to assess proviral integration, quantified using two-step Alu-gag PCR at indicated time points. The values are means (n = 3) and SD. An unpaired t test was used. (E) THP-1-derived macrophages infected with Nef-defective HIV-1 produced as in panel B were treated with DMSO or AZT and used to assess cytoplasmic viral transcript abundance by qPCR at indicated time points. The values are means (n = 3) and SD. An unpaired t test was used. (F) The viral transcript abundance in infected CD4⁺ cells and CD14⁺-derived macrophages quantified using qPCR 24 h postinfection. The values are means (n = 3) and SD. An unpaired t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 4

Infection from SERINC5+ virion leads to the retention of viral RNA in the nucleus. (A) HIV-1 RNA was detected by fluorescent in-situ hybridization (FISH) 20 h postinfection in THP-1 macrophages infected with SERINC5+/− Nef-defective HIV-1. Green puncta indicate viral RNA, and blue represents the Hoechst-stained nuclei. Bar, 10 μm. Right panel, the ratio of puncta in the nucleus to total puncta in infected cells (n = 100). The foci of viral RNA were counted on Fiji-2 software. (B) β-Actin and HIV-1 gag mRNA-remaining (%) quantified by qPCR from the indicated polysome fractions of THP-1-derived macrophages that were infected with SERINC5+/− Nef-defective HIV-1. The values are means (n = 3) and SD. An unpaired t test was used. (C) Western blot indicating intracellular HIV-1 P24 levels and corresponding β-actin from THP-1-derived macrophages infected with SERINC5+/− Nef-defective HIV-1. (D) Cell-free supernatant from the infected THP-1-derived macrophages collected at the indicated time points and subjected to P24 enzyme-linked immunosorbent assay (ELISA). The values are means (n = 3) and SD. An unpaired t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 5

Induction of RPL35 and DRAP1 upon SERINC5 incorporated virion challenge. (A) RNA-sequencing and analysis workflow for scoring differentially expressed genes in the indicated conditions. (B) Differentially expressed genes between HIV-1 (SERINC5−)-infected and HIV-1 (SERINC5+)-infected cells following the RNA-sequencing workflow described in panel A. The values are means (n = 2) and SD. (C) RT-normalized infectivity scored from THP-1 target cells after knockdown of the indicated genes by short hairpin RNAs. Nef-defective SERINC5+/− HIV-1 was produced from HEK293T by cotransfecting pMD2G, NL4-3, and Luciferase Env(−), R(−) Nef(−) along with SERINC5 expressor or empty vector. The values are means (n = 3) and SD. An unpaired t test was used. (D) RPL35 and DRAP1 mRNA expression quantified using qPCR 24 h postinfection from infected CD4⁺ T cells and CD14⁺-derived macrophages from three donors. The values are means (n = 3) and SD. An unpaired t test was used. (E) RPL35 and DRAP1 mRNA expression quantified using qPCR from THP-1-derived macrophages infected with SERINC5+/SERINC5−/SERINC5+Nef-HIV-1 produced from HEK293T. The values are means (n = 3) and SD. An unpaired t test was used. (F) Western blotting of RPL35, DRAP1, and the corresponding β-actin from the THP-1 macrophages infected with SERINC5+/SERINC5−/SERINC5+Nef-HIV-1 produced from HEK293T. Right panel, densitometry plot depicting relative RPL35 and DRAP1 protein expression in infected THP-1 macrophages obtained from Western blots (n = 3). (G) THP-1 cells lacking RPL35, DRAP1, or RPL35 and DRAP1 were generated. Macrophages derived from these cells were infected with Nef-defective HIV-1 produced as in panel C. The infectivity rescue with SERINC5 in GFP knockout (nonrelevant target) THP-1 macrophages was set to 1 for comparison with the other cell lines. The values are means (n = 3) and SD. An unpaired t test was used. Lower panel, Western blot showing RPL35, DRAP1 levels and corresponding β-actin in indicated knockout THP-1 cells. (H) THP-1-derived macrophages (GFP, RPL35, and/or DRAP1 knockouts) were infected with Nef-defective HIV-1 produced as in panel C. Viral transcript abundance in the cytoplasm of infected cells was quantified using qPCR 24 h postinfection. The values are means (n = 3) and SD. An unpaired t test was used. (I) Western blot showing HIV-1 p24 levels and corresponding β-actin from THP-1-derived macrophages infected with Nef-defective HIV-1 produced as in panel C. The values are means (n = 3) and SD. An unpaired t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; WT, wild type.

FIG 6

Analysis of RPL35 and DRAP1 protein interactome. (A) Experimental setup and analysis of liquid chromatography-mass spectrometry (LC-MS) data acquired after immunoprecipitation (IP) of RPL35 and DRAP1. (B) Detection of C-terminally HA-tagged MCE1 and corresponding β-actin from the input lysates and from the RPL35 and DRAP1 immunoprecipitates from infected THP-1 macrophages by Western blotting. THP-1 stably expressing MCE-1-HA were generated by lentiviral transduction and were either mock-infected or infected with HIV-1 SERINC5+/− produced from HEK293T. (C) Reciprocal detection by Western blotting of C-terminally HA-tagged MCE1, RPL35, DRAP1, and the corresponding β-actin from input lysate and MCE1 HA immunoprecipitates obtained from infected MCE1-HA-expressing THP-1 macrophages.

FIG 7

MCE1 and Tat interaction and its effect on viral RNA capping. (A) Detection by Western blotting of HIV-1 Tat and corresponding β-actin from the input lysates and MCE1 HA immunoprecipitates from MCE-1 HA-expressing THP-1 macrophages that were either mock-infected or infected with SERINC5+/− HIV-1 produced by cotransfecting VSV-G, NL4-3, Luciferase Env(−) R(−), SERINC5, or the equivalent empty vector from HEK293T. (B) MCE-1 ChIP assay from MCE1-HA-expressing THP-1 macrophages infected with Nef-defective SERINC5+/− HIV-1 as in panel A depicting Trans-activation response (TAR) region enrichment. The values are means (n = 3) and SD. An unpaired t test was used. (C) Uncapped transcripts quantified using RNA-ligation-mediated reverse transcription qPCR from THP-1 macrophages infected with Nef-defective HIV-1 produced as in panel A. The values are means (n = 3) and SD. An unpaired t test was used. (D) Uncapped transcripts in infected CD4⁺ T cells and CD14⁺-derived macrophages of three donors measured as in panel c. The values are means (n = 3) and SD. An unpaired t test was used. (E) HIV-1 mRNA stability measured by qPCR after actinomycin D treatment at the indicated time points in THP-1 macrophages infected as in panel A. The values are means (n = 3) and SD. An unpaired t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (F) A plausible model for SERINC5-mediated regulation of viral gene expression in macrophages. LTR, long terminal repeat.