Abstract
Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions.
Highlights
Alphaviruses are arthropod-borne viruses that cause frequent epidemics in humans and other vertebrates
By treating infected cells with fusogenic low-pH media, we show that the nascent virions were able to fuse to the plasma membrane (PM) of filopodial extensions of the infected cells, and we provide evidence for the presence of virions on the outside of these filopodia
WmhCehnercryel‐lEs2were treated at pH 4, the fluorescent signals were lost from the fused virions on the filopodia as well as the glycoprotein spikes present on the PM while the fluorescence was retained for the mCherry-E2 present within the cells (Figure 6-4 panels E and F)
Summary
Alphaviruses are arthropod-borne viruses that cause frequent epidemics in humans and other vertebrates. Previous correlative light and electron microscopy studies using fluorescent SINV have provided information about alphavirus budding Such studies established that glycoprotein E2 is enriched on the PM in localized patches that contain other viral structural proteins, from which capsid protein interacts with E2 protein for virus budding. By treating infected cells with fusogenic low-pH media, we show that the nascent virions were able to fuse to the PM of filopodial extensions of the infected cells, and we provide evidence for the presence of virions on the outside of these filopodia This FP-tagged virus can be employed as a tool in high-resolution live and fixed cell imaging coupled with other labeled host proteins and other components to study various aspects of the alphavirus lifecycle
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