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In a previous study to understand how the chikungunya virus (CHIKV) E1 glycoprotein β-strand c functions, we identified several attenuating variants at E1 residue V80 and the emergence of second-site mutations in the fusion loop (E1-M88L) and hinge region (E1-N20Y) with the V80 variants in vivo. The emergence of these mutations led us to question how changes in E1 may contribute to CHIKV infection at the molecular level. Here, we use molecular dynamics to understand how changes in the E1 glycoprotein may influence the CHIKV glycoprotein E1-E2 complex. We found that E1 domain II variants lead to E2 conformational changes, allowing us to hypothesize that emerging variants E1-M88L and E1-N20Y could also change E2 conformation and function. We characterized CHIKV E1-M88L and E1-N20Y in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. We found that CHIKV E1-N20Y enhanced infectivity in mosquito cells, while the CHIKV E1-M88L variant enhanced infectivity in both BHK-21 and C6/36 cells and led to changes in viral cholesterol-dependence. Moreover, we found that E1-M88L and E1-N20Y changed E2 conformation, heparin binding, and interactions with the receptor Mxra8. Interestingly, the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, yet was attenuated in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type-dependent entry. Taken together, these studies show that key residues in the CHIKV E1 domain II and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry. IMPORTANCE Arboviruses are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo to address these gaps in our knowledge. Here, we show that changes in the CHIKV E1 domain II and hinge alter E2 conformations leading to changes in virus-receptor and -glycosaminoglycan interactions and cell-specific infection. These results highlight that adaptive changes in E1 can have a major effect on virus attachment and entry, furthering our knowledge of how alphaviruses infect mammals and insects. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like other coronaviruses, replicates their genome in virus-induced cytosolic membrane-bound replication organelles (ROs). SARS-CoV-2 promotes the biogenesis of ROs by inducing the rearrangement of endoplasmic reticulum (ER) membranes. NSP3, NSP4, and NSP6 are transmembrane viral non-structural proteins (NSPs) and essential players in the formation of ROs. To understand how these three NSPs work synergistically with host-binding proteins, we performed affinity purifications followed by mass spectrometry analyses to study the host-viral protein-protein interactome of NSP3, NSP4, and NSP6 expressed individually and in combination. Through this analysis, we identified two host transmembrane proteins, REEP5 and TRAM1, as critical interacting partners of NSP3 that localize at the membrane of the RO. REEP5 interacts with TRAM1 endogenously and binds NSP3 during SARS-CoV-2 infection. REEP5 knockout reduces ER membrane rearrangements and inhibits SARS-CoV-2 replication. Collectively, our study shows that the host REEP5/TRAM1 complex binds NSP3, promoting RO biogenesis and viral replication. IMPORTANCE Generation of virus-host protein–protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in β-strand c (V114M) of domain II. We have previously shown that the analogous b-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E b-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo. [ABSTRACT FROM AUTHOR]

Arthropod-borne viruses represent a significant public health threat worldwide yet there are few antiviral therapies or prophylaxis targeting these pathogens. In particular, the development of novel antivirals for high-risk populations such as pregnant women is essential to prevent devastating disease such as that which was experienced with the recent outbreak of Zika virus ZIKV) in the Americas. One potential avenue to identify new and pregnancy-acceptable antiviral compounds is to repurpose well-known and widely used FDA approved drugs. In this study, we addressed the antiviral role of atovaquone, a FDA Pregnancy Category C drug and pyrimidine biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and that this antiviral effect occurred early during infection at the initial steps of viral RNA replication. Moreover, we were able to complement viral replication and virion production with the addition of exogenous pyrimidine nucleosides indicating that atovaquone is functioning through the inhibition of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent manner providing evidence that atovaquone may function as an antiviral in humans. Taken together, these studies suggest that atovaquone could be a broad- spectrum antiviral drug and a potential attractive candidate for the prophylaxis or treatment of arbovirus infection in vulnerable populations, such as pregnant women and children. Importance The ability to protect vulnerable populations such as pregnant women and children from Zika virus and other arbovirus infections is essential to preventing the devastating complications induced by these viruses. One class of antiviral therapies may lie in known pregnancy- acceptable drugs that have the potential to mitigate arbovirus infections and disease yet this has not been explored in detail. In this study, we show that the common antiparasitic drug, atovaquone, inhibits arbovirus replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo human placental explant model. Our study provides a novel function for atovaquone and highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can be the key to better addressing the immediate need for treating viral infections and preventing potential birth complications and future disease. [ABSTRACT FROM AUTHOR]