Abstract
ABSTRACTAlphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. The virus membrane is covered by a lattice composed of 80 spikes, each a trimer of heterodimers of the E2 and E1 transmembrane proteins. During virus endocytic entry, the E1 glycoprotein mediates the low-pH-dependent fusion of the virus membrane with the endosome membrane, thus initiating virus infection. While much is known about E1 structural rearrangements during membrane fusion, it is unclear how the E1/E2 dimer dissociates, a step required for the fusion reaction. A recent Alphavirus cryo-electron microscopy reconstruction revealed a previously unidentified D subdomain in the E2 ectodomain, close to the virus membrane. A loop within this region, here referred to as the D-loop, contains two highly conserved histidines, H348 and H352, which were hypothesized to play a role in dimer dissociation. We generated Semliki Forest virus mutants containing the single and double alanine substitutions H348A, H352A, and H348/352A. The three D-loop mutations caused a reduction in virus growth ranging from 1.6 to 2 log but did not significantly affect structural protein biosynthesis or transport, dimer stability, virus fusion, or specific infectivity. Instead, growth reduction was due to inhibition of a late stage of virus assembly at the plasma membrane. The virus particles that are produced show reduced thermostability compared to the wild type. We propose the E2 D-loop as a key region in establishing the E1-E2 contacts that drive glycoprotein lattice formation and promote Alphavirus budding from the plasma membrane.
Highlights
Alphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses
We propose that H348 and H352 are critical for the formation of the E1/E2 lattice at the plasma membrane, highlighting the importance of glycoprotein interactions in virus budding
While some Alphavirus mutants with impaired virus production are rescued by growth at 28°C [25, 26], the growth of the H348A, H352A, and H348/352A mutants remained inhibited when RNA-electroporated BHK cells were incubated at 28°C (Fig. 2B)
Summary
Alphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. We define the role of the E2 protein juxtamembrane D-loop, which contains highly conserved histidine residues at positions 348 and 352 These histidines do not play an important role in virus fusion and infection. Alphaviruses are spherical particles with very organized structures and high specific infectivity (reviewed in references 1, 8, and 9) Both the capsid and envelope proteins are arranged with Tϭ4 icosahedral symmetry. The internal nucleocapsid core contains a single copy of the RNA genome surrounded by a lattice of 240 copies of the capsid protein This core is enveloped by the virus lipid bilayer, which contains a lattice composed of 240 copies of the transmembrane E1 and E2 proteins, closely associated as heterodimers and further organized into 80 trimeric spikes. The nucleocapsid is thereby released into the cytoplasm, where it dissociates to release the viral RNA for subsequent translation and replication
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