Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine.

T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of Chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce Chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process that appears to be mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production.

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.

Temperate Conditions Limit Zika Virus Genome Replication.

Zika virus is a mosquito-borne flavivirus known to cause severe birth defects and neuroimmunological disorders. We have previously demonstrated that mosquito transmission of Zika virus decreases with temperature. While transmission was optimized at 29°C, it was limited at cool temperatures (<22°C) due to poor virus establishment in the mosquitoes. Temperature is one of the strongest drivers of vector-borne disease transmission due to its profound effect on ectothermic mosquito vectors, viruses, and their interaction. Although there is substantial evidence of temperature effects on arbovirus replication and dissemination inside mosquitoes, little is known about whether temperature affects virus replication directly or indirectly through mosquito physiology. In order to determine the mechanisms behind temperature-induced changes in Zika virus transmission potential, we investigated different steps of the virus replication cycle in mosquito cells (C6/36) at optimal (28°C) and cool (20°C) temperatures. We found that the cool temperature did not alter Zika virus entry or translation, but it affected genome replication and reduced the amount of double-stranded RNA replication intermediates. If replication complexes were first formed at 28°C and the cells were subsequently shifted to 20°C, the late steps in the virus replication cycle were efficiently completed. These data suggest that cool temperature decreases the efficiency of Zika virus genome replication in mosquito cells. This phenotype was observed in the Asian lineage of Zika virus, while the African lineage Zika virus was less restricted at 20°C. IMPORTANCE With half of the human population at risk, arboviral diseases represent a substantial global health burden. Zika virus, previously known to cause sporadic infections in humans, emerged in the Americas in 2015 and quickly spread worldwide. There was an urgent need to better understand the disease pathogenesis and develop therapeutics and vaccines, as well as to understand, predict, and control virus transmission. In order to efficiently predict the seasonality and geography for Zika virus transmission, we need a deeper understanding of the host-pathogen interactions and how they can be altered by environmental factors such as temperature. Identifying the step in the virus replication cycle that is inhibited under cool conditions can have implications in modeling the temperature suitability for arbovirus transmission as global environmental patterns change. Understanding the link between pathogen replication and environmental conditions can potentially be exploited to develop new vector control strategies in the future.

Ebola Virus Requires Phosphatidylserine Scrambling Activity for Efficient Budding and Optimal Infectivity.

Ebola virus (EBOV) attaches to target cells using two categories of cell surface receptors: C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic debris. Many enveloped viruses also contain exposed PS and can therefore exploit these receptors for cell entry. Viral infection can induce PS externalization in host cells, resulting in increased outer PS levels on budding virions. Scramblase enzymes carry out cellular PS externalization; thus, we targeted these proteins in order to manipulate viral envelope PS levels. We investigated two scramblases previously identified to be involved in EBOV PS levels, transmembrane protein 16F and Xk-related protein 8 (XKR8), as possible mediators of cellular and viral envelope surface PS levels during the replication of recombinant vesicular stomatitis virus containing its native glycoprotein (rVSV/G) or the EBOV glycoprotein (rVSV/EBOV-GP). We found that rVSV/G and rVSV/EBOV-GP virions produced in XKR8 knockout cells contain decreased levels of PS on their surfaces, and the PS-deficient rVSV/EBOV-GP virions are 70% less efficient at infecting cells through PS receptors. We also observed reduced rVSV and EBOV virus-like particle (VLP) budding in ΔXKR8 cells. Deletion of XKR8 in HAP1 cells reduced rVSV/G and rVSV/EBOV-GP budding by 60 and 65%, respectively, and reduced Ebola VLP budding more than 60%. We further demonstrated that caspase cleavage of XKR8 is required to promote budding. This suggests that XKR8, in addition to mediating virion PS levels, may also be critical for enveloped virus budding at the plasma membrane. IMPORTANCE Within the last decade, countries in western and central Africa have experienced the most widespread and deadly Ebola outbreaks since Ebola virus was identified in 1976. While outbreaks are primarily attributed to zoonotic transfer events, new evidence is emerging outbreaks may be caused by a combination of spillover events and viral latency or persistence in survivors. The possibility that Ebola virus can remain dormant and then reemerge in survivors highlights the critical need to prevent the virus from entering and establishing infection in human cells. Thus far, host cell scramblases TMEM16F and XKR8 have been implicated in Ebola envelope surface phosphatidylserine (PS) and cell entry using PS receptors. We assessed the contributions of these proteins using CRISPR knockout cells and two EBOV models: rVSV/EBOV-GP and EBOV VLPs. We observed that XKR8 is required for optimal EBOV envelope PS levels and infectivity and particle budding across all viral models.