Capsid-deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane

Abstract

Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.

Why Enveloped Viruses Need Cores—The Contribution of a Nucleocapsid Core to Viral Budding

“…It is worth considering this observation in the context of recent experimental observations on budding in the absence of an NC, as well as experimental measurements on viral particle elastic properties. Several experimental studies have reported GP-directed budding from cells in which the NC proteins are impaired (18,19). In particular, Ruiz-Guillen et al (19) recently showed that cells expressing the genome and GPs, but not the capsid protein, for Sindbis and Semliki Forest virus generate infectious viral particles that can propagate in mammalian cells.…”

“…Several experimental studies have reported GP-directed budding from cells in which the NC proteins are impaired (18,19). In particular, Ruiz-Guillen et al (19) recently showed that cells expressing the genome and GPs, but not the capsid protein, for Sindbis and Semliki Forest virus generate infectious viral particles that can propagate in mammalian cells. For the wild-type virus, in which the GPs assemble around the NC, they estimated a virion diameter of 60-70 nm, whereas the GP-only particles were typically 100-150 nm.…”

“…However, several studies have challenged this conclusion. In particular, Forsell et al (18) reported successful assembly and budding of alphavirus despite mutations that inhibited NC assembly by impairing interactions between NC proteins, whereas Ruiz-Guillen et al (19) observed budding of infectious alphavirus particles from cells that did not express the capsid gene. In both cases, infectious particles were assembled and released or budded from the cell.…”

“…In this article, we perform molecular dynamics simulations on a coarse-grained model for GPs, the NC, and a lipid bilayer membrane to elucidate the forces driving enveloped virus budding. Our model is motivated by the alphavirus structure and experimental observations on alphavirus budding (12,18,19), but we consider our results in the broader context of enveloped viruses. To evaluate the relative roles of a preassembled NC compared to the assembly of transmembrane glycoproteins in driving budding, we perform two sets of simulations.…”

“…1 a) by including only the membrane and model GPs, whose geometry and interactions drive formation of an icosahedral shell with the geometry of the alphavirus envelope. This model directly applies to experiments on budding from cells in which capsid assembly was eliminated (18,19). The second set of simulations includes model GPs and a preassembled NC, thus allowing for NC-directed budding ( Fig.…”