FAM46C Is an Interferon-Stimulated Gene That Inhibits Lentiviral Particle Production by Modulating Autophagy.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity.

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.

Heterogeneity of Latency Establishment in the Different Human CD4+ T Cell Subsets Stimulated with IL-15.

HIV integrates into the host genome, creating a viral reservoir of latently infected cells that persists despite effective antiretroviral treatment. CD4-positive (CD4+) T cells are the main contributors to the HIV reservoir. CD4+ T cells are a heterogeneous population, and the mechanisms of latency establishment in the different subsets, as well as their contribution to the reservoir, are still unclear. In this study, we analyzed HIV latency establishment in different CD4+ T cell subsets stimulated with interleukin 15 (IL-15), a cytokine that increases both susceptibility to infection and reactivation from latency. Using a dual-reporter virus that allows discrimination between latent and productive infection at the single-cell level, we found that IL-15-treated primary human CD4+ T naive and CD4+ T stem cell memory (TSCM) cells are less susceptible to HIV infection than CD4+ central memory (TCM), effector memory (TEM), and transitional memory (TTM) cells but are also more likely to harbor transcriptionally silent provirus. The propensity of these subsets to harbor latent provirus compared to the more differentiated memory subsets was independent of differential expression of pTEFb components. Microscopy analysis of NF-κB suggested that CD4+ T naive cells express smaller amounts of nuclear NF-κB than the other subsets, partially explaining the inefficient long terminal repeat (LTR)-driven transcription. On the other hand, CD4+ TSCM cells display similar levels of nuclear NF-κB to CD4+ TCM, CD4+ TEM, and CD4+ TTM cells, indicating the availability of transcription initiation and elongation factors is not solely responsible for the inefficient HIV gene expression in the CD4+ TSCM subset. IMPORTANCE The formation of a latent reservoir is the main barrier to HIV cure. Here, we investigated how HIV latency is established in different CD4+ T cell subsets in the presence of IL-15, a cytokine that has been shown to efficiently induce latency reversal. We observed that, even in the presence of IL-15, the less differentiated subsets display lower levels of productive HIV infection than the more differentiated subsets. These differences were not related to different expression of pTEFb, and modest differences in NF-κB were observed for CD4+ T naive cells only, implying the involvement of other mechanisms. Understanding the molecular basis of latency establishment in different CD4+ T cell subsets might be important for tailoring specific strategies to reactivate HIV transcription in all the CD4+ T subsets that compose the latent reservoir.

Anti-spike antibodies and neutralising antibody activity in people living with HIV vaccinated with COVID-19 mRNA-1273 vaccine: a prospective single-centre cohort study.

BACKGROUND: Vaccines against COVID-19 are a powerful tool to control the current SARS-CoV-2 pandemic. A thorough description of their immunogenicity among people living with HIV (PLWHIV) is necessary. We aimed to assess the immunogenicity of the mRNA-1273 vaccine among PLWHIV. METHODS: In this prospective cohort, adult PLWHIV outpatients were enrolled during the Italian vaccination campaign. Enrolment was allowed irrespective of ongoing combination antiretroviral therapy (ART), plasma HIV viral load and CD4+ T cell count. A two-dose regimen of mRNA-1273, with administrations performed 28 days apart, was employed. The primary outcomes were anti-spike (anti-S) antibody titres and neutralising antibody activity, assessed 28 days after completing the vaccination schedule. A convenient sample of individuals not affected by HIV was also collected to serve as control (referred as healthy-donors, HDs). FINDINGS: We enrolled 71 PLWHIV, mostly male (84·5%), with a mean age of 47 years, a median CD4+ T cell count of 747·0 cells per µL and a median HIV viral load <50 copies/mL. COVID-19-experienced PLWHIV displayed higher anti-S antibody titres (p=0·0007) and neutralising antibody activity in sera (p=0·0007) than COVID-19-naïve PLWHIV. When stratified according to CD4+ T cell count (<350 cells/µL, 350-500 cells/µL, >500 cells/µL), anti-S antibody titres (6/71, median 2173 U/mL [IQR 987-4109]; 7/71, 5763 IU/mL [IQR 4801->12500]; 58/71, 2449 U/mL [IQR 1524-5704]) were not lower to those observed among HDs (10, median 1425 U/mL [IQR 599-6131]). In addition, neutralising antibody activity, stratified according to the CD4+ T cell count (6/71, median 1314 [IQR 606-2477]; 7/71, 3329 IU/mL [IQR 1905-10508]; 58/71, 1227 U/mL [IQR 761-3032]), was like those displayed by HDs (10, median 2112 U/mL [IQR 719-8889]). INTERPRETATION: In our cohort of PLWHIV with well-controlled ART, stable viral suppression and robust CD4+ T cell count, inoculation with mRNA-1273 vaccine given 4 weeks apart produced detectable humoral immune response, similar to individuals without HIV infection, supporting vaccination in PLWHIV. FUNDING: This study was partially supported by Italian Ministry of Health Ricerca Corrente 2021, by Intesa San Paolo COVID-19 emergency 2020 funds, and by Fondazione Cariplo Grant (INNATE-CoV).