SUMMARY Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections by inhibiting virus-cell fusion. IFITM proteins also inhibit HIV-1 replication through mechanisms only partially understood. We show that when expressed in uninfected lymphocytes, IFITM proteins exert protective effects during cell-free virus infection, but this restriction can be overcome upon HIV-1 cell-to-cell spread. However, when present in virus-producing lymphocytes, IFITM proteins colocalize with viral Env and Gag proteins and incorporate into nascent HIV-1 virions to limit entry into new target cells. IFITM in viral membranes is associated with impaired virion fusion, offering additional and more potent defense against virus spread. Thus, IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses.

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S ‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxΦ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3-dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin-mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion.

Summary APOBEC3G (A3G) is a host cytidine deaminase that inhibits retroviruses. HIV and related primate lentiviruses encode Vif, which counteracts A3G by inducing its degradation. This Vif-mediated A3G inhibition is species-specific, suggesting that the A3G-Vif interaction has evolved as primate lentiviruses have adapted to their hosts. We examined the evolutionary dynamics of the A3G-Vif interaction within four African Green Monkey (AGM) subspecies, which are each naturally infected with a distinct simian immunodeficiency virus (SIV). We identified single amino acid changes within A3G in two AGM subspecies that render it resistant to Vif proteins, except for Vif from the viruses that naturally infect these subspecies. Moreover, experimental infection of AGMs shows that Vif can rapidly adapt to these arising Vif-resistant A3G genotypes. These data suggest that despite being generally non-pathogenic in its natural host, SIV infection selects for Vif-resistant forms of A3G in AGM populations, driving Vif counter-evolution and functional divergence.

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.

Naturally circulating lentiviruses are abundant in African primate species today, yet their origins and history of transmitting between hosts remain obscure. As a means to better understand the age of primate lentiviruses, we analyzed primate genomes for signatures of lentivirus-driven evolution. Specifically, we studied the adaptive evolution of host restriction factor APOBEC3G (A3G) in Old World Monkey (OWM) species. We find recurrent mutation of A3G in multiple primate lineages at sites that determine susceptibility to antagonism by the lentiviral accessory protein Vif. Using a broad panel of SIV Vif isolates, we demonstrate that natural variation in OWM A3G confers resistance to Vif-mediated degradation, suggesting that adaptive variants of the host factor were selected upon exposure to pathogenic lentiviruses at least 5–6 million years ago (MYA). Furthermore, in members of the divergent Colobinae subfamily of OWM, a multi-residue insertion event in A3G that arose at least 12 MYA blocks the activity of Vif, suggesting an even more ancient origin of SIV. Moreover, analysis of the lentiviruses associated with Colobinae monkeys reveal that the interface of the A3G-Vif interaction has shifted and given rise to a second genetic conflict. Our analysis of virus-driven evolution describes an ancient yet ongoing genetic conflict between simian primates and lentiviruses on a million-year time scale.

The interferon-induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV-1 virions and inhibit virus fusion and cell-to-cell spread, with IFITM3 showing the greatest potency. Here, we report that amino-terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. This suggests that "runaway" IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade-off in antiviral specificity, as variants exhibiting enhanced activity against HIV-1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.