Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. It remains a naturally emerging disease as well as a highly developed biological weapon. VEEV is transmitted to humans in nature by mosquito vectors. Little is known about VEEV entry, especially in mosquito cells. Here, a novel luciferase-based virus entry assay is used to show that the entry of VEEV into mosquito cells requires acidification. Furthermore, mosquito homologs of key human proteins (Rab5 and Rab7) involved in endocytosis were isolated and characterized. Rab5 is found on early endosomes and Rab7 on late endosomes and both are important for VEEV entry in mammalian cells. Each was shown to have analogous function in mosquito cells to that seen in mammalian cells. The wild-type, dominant negative and constitutively active mutants were then used to demonstrate that VEEV requires passage through early and late endosomes before infection can take place. This work indicates that the infection mechanism in mosquitoes and mammals is through a common and ancient evolutionarily conserved pathway.

Massive infection of memory CD4 T cells is a hallmark of early simian immunodeficiency virus (SIV) infection, with viral infectionHere we show that, at day 10 p.i., Lin ؊ HLA-DR ؉ CD11c/123 ؊ CD13 ؉ CD14 ؊ macrophages in the jejunal mucosa were infected, albeit at lower levels than CD4 memory T cells. Interestingly, Lin ؊ HLA-DR ؉ CD11c/123 ؊ CD13 ؉ CD14 ؊ macrophages in peripheral blood, like their mucosal counterparts, were preferentially infected compared to Lin ؊ HLA-DR ؉ CD11c/123 ؊ CD13 ؉ CD14 ؉ monocytes, suggesting that differentiated macrophages were selectively infected by SIV. CD13 ؉ CD14 ؊ macrophages expressed low levels of CD4 compared to CD4 T cells but expressed similar levels of CCR5 as lymphocytes. Interestingly, CD13 ؉ CD14 ؊ macrophages expressed Apobec3G at lower levels than CD13 ؉ CD14 ؉ monocytes, suggesting that intracellular restriction may contribute to the differential infection of mononuclear subsets. Taken together, our results suggest that CD13 ؉ CD14 ؊ macrophages in mucosal and peripheral tissues are preferentially infected very early during the course of SIV infection.

One of the most important steps in a productive viral infection is when the virus fuses to a cell membrane and delivers its genome into the cell cytosol. This dynamic event is mediated by interactions between specific virus envelope proteins with their cell-bound receptors. This process is exemplified by Moloney murine leukemia virus (Mo-MLV) where envelope protein interaction with its receptor, mCAT-1, leads to virus-cell membrane fusion and infection of cells. Here, fluorescent nanoparticles (NPs) were coated with Mo-MLV derived membranes (Mo-NPs) by extrusion. Electron microscopy and biochemical analysis showed tight association of the virus membranes and NPs. The coated NPs mimic native virus by binding and entering only cells expressing the virus receptor. Confocal microscopy revealed that the coated NPs were taken up into endocytic compartments containing receptor and were also seen associated with caveolin, a marker of caveolae. To demonstrate that the Mo-NPs could escape endosomes and deliver a protein cargo into the cell cytosol, beta-lactamase (betalac) was covalently coupled to the Mo-NP cores and incubated with cells. betalac activity was only detected in the cytosol of mCAT-1-expressing cells. This is the first time that virus proteins have been used to specifically target NPs to receptor-bearing cells as well as penetration into the cell cytosol. Extrusion provides a rapid, detergent-free method to couple virus membranes to NPs and should be readily applicable for many other virus and NP types.

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