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Abstract HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CANTD-CANTD inter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal β-hairpin. (3) Altered conformations of E45Aa α9-helices compared to WT, E45A/R132T, WTPF74, WTNup153, and WTCPSF6 decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be responsible for SER5’s antiviral activity. This and other reports that document modulation of HIV-1 infectivity by viral lipid composition prompted us to investigate the role of PS in regulating SER5-mediated HIV-1 restriction. First, we show that the level of SER5 incorporation into virions correlates with an increase in PS levels in the outer leaflet of the viral membrane. We developed an assay to estimate the PS distribution across the viral membrane and found that SER5, but not SER2, which lacks antiviral activity, abrogates PS asymmetry by externalizing this lipid. Second, SER5 incorporation diminished the infectivity of pseudoviruses produced from cells lacking a flippase subunit CDC50a and, therefore, exhibited a higher baseline level of surface-accessible PS. Finally, exogenous manipulation of the viral PS levels utilizing methyl-alpha-cyclodextrin revealed a lack of correlation between external PS and virion infectivity. Taken together, our study implies that the increased PS exposure to SER5-containing virions itself is not directly linked to HIV-1 restriction.

Host proteins CPSF6, NUP153, and SEC24C are vital for HIV-1 infection. They bind to the viral capsid protein and contribute to shuttling of virions through the cytoplasm (SEC24C), import into the nucleus (NUP153 and CPSF6) and subsequent trafficking to preferred integration sites (CPSF6). Here, Wei et al. combine structural, biochemical and virological assays to emphasize the importance of prion-like low complexity domains surrounding short phenylalanine-glycine regions in binding and increasing the avidity when interacting with viral capsid.

Human interferon-induced transmembrane (IFITM) proteins inhibit the fusion of a broad spectrum of enveloped viruses, both when expressed in target cells and when present in infected cells. Upon expression in infected cells, IFITMs incorporate into progeny virions and reduce their infectivity by a poorly understood mechanism. Since only a few envelope glycoproteins (Envs) are present on HIV-1 particles, and Env clustering has been proposed to be essential for optimal infectivity, we asked if IFITM protein incorporation modulates HIV-1 Env clustering. The incorporation of two members of the IFITM family, IFITM1 and IFITM3, into HIV-1 pseudoviruses correlated with a marked reduction of infectivity. Super-resolution imaging of Env distribution on single HIV-1 pseudoviruses did not reveal significant effects of IFITMs on Env clustering. However, IFITM3 reduced the Env processing and incorporation into virions relative to the control and IFITM1-containing viruses. These results show that, in addition to interfering with the Env function, IFITM3 restricts HIV-1 Env cleavage and incorporation into virions. The lack of notable effect of IFITMs on Env clustering supports alternative restriction mechanisms, such as modification of the properties of the viral membrane.

Interferon-induced transmembrane proteins (IFITMs) block the fusion of diverse enveloped viruses, likely through increasing the cell membrane’s rigidity. Previous studies have reported that the antiviral activity of the IFITM family member, IFITM3, is antagonized by cell pretreatment with rapamycin derivatives and cyclosporines A and H (CsA and CsH) that promote the degradation of IFITM3. Here, we show that CsA and CsH potently enhance virus fusion with IFITM1- and IFITM3-expressing cells by inducing their rapid relocalization from the plasma membrane and endosomes, respectively, towards the Golgi. This relocalization is not associated with a significant degradation of IFITMs. Although prolonged exposure to CsA induces IFITM3 degradation in cells expressing low endogenous levels of this protein, its levels remain largely unchanged in interferon-treated cells or cells ectopically expressing IFITM3. Importantly, the CsA-mediated redistribution of IFITMs to the Golgi occurs on a much shorter time scale than degradation and thus likely represents the primary mechanism of enhancement of virus entry. We further show that rapamycin also induces IFITM relocalization toward the Golgi, albeit less efficiently than cyclosporines. Our findings highlight the importance of regulation of IFITM trafficking for its antiviral activity and reveal a novel mechanism of the cyclosporine-mediated modulation of cell susceptibility to enveloped virus infection.

Serine incorporator 5 (SER5) is a protein that upon incorporation into virions inhibits HIV-1 infectivity by interfering with the ability of the Env glycoprotein to promote viral fusion. The mechanisms by which SER5 antagonizes HIV-1 fusion are not well understood. A recent study of SER5’s structure revealed a lipid-binding pocket, suggesting the ability to sequester lipids. This finding, along with the well-documented modulation of HIV-1 infectivity by viral lipids, especially cholesterol, prompted our examination of SER5′s effect on the general lipid order of the HIV-1 membrane. Pseudoviruses bearing the SER5-sensitive HXB2-Env and containing SER5 or SER2, a control protein that lacks antiviral activity, were analyzed using two distinct lipid-order probes. We show that SER5 incorporation does not noticeably affect the lipid order of pseudoviruses. Although viral cholesterol extraction reduces HIV-1 infectivity, SER5+ viruses are less sensitive to cholesterol extraction than the control samples. In contrast, the virus’ sensitivity to cholesterol oxidation was not affected by SER5 incorporation. The hydrolytic release of sphingomyelin-sequestered cholesterol had a minimal impact on the apparent resistance to cholesterol extraction. Based on these results, we propose that a subpopulation of more stable Env glycoproteins responsible for the residual infectivity of SER5+ viruses is less sensitive to the cholesterol content of the viral membrane.

SERINC5 incorporates into HIV-1 particles and inhibits the ability of Env glycoprotein to mediate virus-cell fusion. SERINC5-resistance maps to Env, with primary isolates generally showing greater resistance than laboratory-adapted strains. Here, we examined a relationship between the inhibition of HIV-1 infectivity and the rate of Env inactivation using a panel of SERINC5-resistant and -sensitive HIV-1 Envs. SERINC5 incorporation into pseudoviruses resulted in a faster inactivation of sensitive compared to resistant Env strains. A correlation between fold reduction in infectivity and the rate of inactivation was also observed for multiple Env mutants known to stabilize and destabilize the closed Env structure. Unexpectedly, most mutations disfavoring the closed Env conformation rendered HIV-1 less sensitive to SERINC5. In contrast, functional inactivation of SERINC5-containing viruses was significantly accelerated in the presence of a CD4-mimetic compound, suggesting that CD4 binding sensitizes Env to SERINC5. Using a small molecule inhibitor that selectively targets the closed Env structure, we found that, surprisingly, SERINC5 increases the potency of this compound against a laboratory-adapted Env which prefers a partially open conformation, indicating that SERINC5 may stabilize the closed trimeric Env structure. Our results reveal a complex effect of SERINC5 on Env conformational dynamics that promotes Env inactivation and is likely responsible for the observed restriction phenotype.

Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry.