Mechanisms of alphavirus membrane fusion and assembly
Alphaviruses are enveloped RNA arboviruses that include important human pathogens. These viruses cause serious and debilitating diseases, with recent outbreaks resulting in millions of cases worldwide. There are currently no licensed vaccines or treatments.;Alphavirus infection involves uptake via clathrin-mediated endocytosis and viral envelope fusion with the host endosome membrane. Fusion is triggered by endosomal low pH and mediated by the membrane fusion protein, El. El and its companion protein, E2, are present on the virus envelope as heterodimers that are further assembled into 80 trimeric spikes. Structural studies revealed a previously unidentified subdomain D in the membrane-proximal region of E2, containing a looped structure termed the D-loop. It was hypothesized that two highly conserved D-loop histidines, H348 and H352, would promote dissociation of El and E2 when protonated at acidic pH. We characterized the role of H348 and H352 using site-directed mutagenesis of the alphavirus Semliki Forest virus (SFV), creating single and double alanine substitutions. Our results indicate a 1.6-2-log reduction in growth for all 3 mutants but at most a modest effect on the pH dependence of fusion and specific infectivity. However, we discovered a novel assembly defect that inhibits the formation of mutant virus particles. Transmission electron microscopy revealed mutant nucleocapsids that were localized at the plasma membrane but failed to bud. Those particles that did successfully bud were of normal morphology, but had a significant reduction in thermostability in comparison to wild type. We propose that H348 and H352, in conjunction with several El residues we identified by structural analysis, mediate formation of the glycoprotein lattice necessary for budding.;Additionally, selection of H348/352A revertant viruses revealed several mutations in the El, E2, and capsid proteins. Two revertant viruses were extensively characterized, E2-T402K and El-T317I C-D180N. These mutations occurred in a region of El known to have inter-spike contacts, and in the cytoplasmic domain of E2 in the region responsible for binding capsid. E2-T402K and El-T317I were both able to partially restore H348/352A growth and assembly, but not virus thermostability. Of interest, CD180N strongly decreased growth in the H348/352A background. These results further support the role of the D-loop in membrane glycoprotein interactions and describes an additional role of the D-loop in mediating downstream E2-capsid binding.;Lastly, we investigated an Aedes albopictus permissive Chikungunya field mutant, E2-L2I0Q. This mutation occurs in conjunction with a primary mutation, El -A226V, and increases virus infectivity in a new mosquito vector, A. albopictus. Due to the location of the E2-L210 residue near the acid sensitive region of E2, we investigated the pH dependence of fusion and of receptor binding as possible mechanisms of increased infectivity. While El-A226V E2-L210Q VLPs had a slight decrease in the pH threshold of fusion, this was attributed to the El-A226V mutation. In addition, the pH of E2L210Q binding was not significantly altered. Thus, we propose that pH dependence is not the mechanism of increased E2-L210Q infectivity, and that E2-L210Q likely mediates increased receptor affinity in A. albopictus.
Source: Dissertation Abstracts International, Volume: 79-03(E), Section: B.;Advisors: Margaret Kielian.