Disruption of Transmembrane Phosphatidylserine Asymmetry by HIV-1 Incorporated SERINC5 Is Not Responsible for Virus Restriction.

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.

Chikungunya virus entry and infectivity is primarily facilitated through cell line dependent attachment factors in mammalian and mosquito cells.

Chikungunya virus (CHIKV) is the causative agent of the human disease chikungunya fever, characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals are currently available for CHIKV. Therefore, the prevention of attachment of viral particles to host cells is a potential intervention strategy. As an arbovirus, CHIKV infects a wide variety of cells in both its mammalian and mosquito host. This broad cell tropism might stem from CHIKV's ability to bind to a variety of entry factors in the host cell including phosphatidylserine receptors (PSRs), glycosaminoglycans (GAGs), and the proteinaceous receptor Mxra8, among others. In this study, we aimed to determine the relevance of each attachment factor during CHIKV entry into a panel of mammalian and mosquito cells. Our data suggest that the importance of particular binding factors during CHIKV infection is highly cell line dependent. Entry into mammalian Vero cells was mediated through attachment to PSRs, mainly T-cell immunoglobulin mucin domain-1 (TIM-1). Conversely, CHIKV infection into HAP1 and NIH3T3 was predominantly mediated by heparan sulfate (HS) and Mxra8, respectively. Entry into mosquito cells was independent of PSRs, HS, and Mxra8. Although entry into mosquito cells remains unclear, our data denotes the importance of careful evaluation of reagents used to identify receptor use in invertebrate cells. While PSRs, GAGs, and Mxra8 all enhance entry in a cell line dependent manner, none of these factors are necessary for CHIKV entry, suggesting additional host factors are involved.

Untargeted Lipidomics of Vesicular Stomatitis Virus-Infected Cells and Viral Particles.

The viral lifecycle is critically dependent upon host lipids. Enveloped viral entry requires fusion between viral and cellular membranes. Once an infection has occurred, viruses may rely on host lipids for replication and egress. Upon exit, enveloped viruses derive their lipid bilayer from host membranes during the budding process. Furthermore, host lipid metabolism and signaling are often hijacked to facilitate viral replication. We employed an untargeted HILIC-IM-MS lipidomics approach and identified host lipid species that were significantly altered during vesicular stomatitis virus (VSV) infection. Many glycerophospholipid and sphingolipid species were modified, and ontological enrichment analysis suggested that the alterations to the lipid profile change host membrane properties. Lysophosphatidylcholine (LPC), which can contribute to membrane curvature and serve as a signaling molecule, was depleted during infection, while several ceramide sphingolipids were augmented during infection. Ceramide and sphingomyelin lipids were also enriched in viral particles, indicating that sphingolipid metabolism is important during VSV infection.

SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To explore S requirements for entry under BSL2 conditions, S has been pseudotyped onto vesicular stomatitis virus (VSV) or retroviral particles with varied success. Several alterations to S were demonstrated to improve pseudoparticle titers, but they have not been systematically compared. In this study, we produced pseudotyped VSV particles with multiple modifications to S, including truncation, mutation, and tagging strategies. The main objective of this study was to determine which modifications of the S protein optimize cell surface expression, incorporation into pseudotyped particles, and pseudoparticle entry. Removal of the last 19 residues of the cytoplasmic tail produced a hyper-fusogenic S, while removal of 21 residues increased S surface production and VSV incorporation. Additionally, we engineered a replication-competent VSV (rVSV) virus to produce the S-D614G variant with a truncated cytoplasmic tail. While the particles can be used to assess S entry requirements, the rVSV∆G/SMet1D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio).