Ribonuclease Sensitivity of Semliki Forest Virus Nucleocapsids

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations s1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of >1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.

Section: Resultssupporting

confidence: 69%

“…The nucleocapsids of alphaviruses are unique in that the RNA and protein condense into a tightly packed but RNasesensitive spherical structure in the absence of the virus envelope (1,11,51). The condensation reaction is highly specific and extremely rapid (50).…”

mentioning

confidence: 99%

Form-determining functions in Sindbis virus nucleocapsids: nucleosomelike organization of the nucleocapsid

Coombs

Brown

1989

“…X-ray crystallography of Sindbis virus, an alphavirus closely related to SFV, has revealed that the C protein differs from other known viral C proteins in having a structure similar to that of chymotrypsin (3). The shell formed by C proteins around the viral RNA has holes, so that the RNA is accessible to small molecules such as RNase (1,16). However, these holes are insufficient for the viral RNA to be accessed and translated by ribosomes.…”

mentioning

confidence: 99%

Role of ribosomes in Semliki Forest virus nucleocapsid uncoating

Singh

Helenius

1992

113

The mechanism by which Semliki Forest virus nucleocapsids are uncoated was analyzed in living cells and in vitro. In BHK-21 cells, uncoating occurred with virtually complete efficiency within 1 to 2 min after the nucleocapsids entered the cytoplasm. It was inhibited by monensin, which blocks nucleocapsid penetration from endosomes. As previously shown for Sindbis virus (G. Wengler and G. Wengler, Virology 134:435-442, 1984), the capsid proteins from incoming nucleocapsids became associated with ribosomes. The ribosomebound capsid proteins were distributed throughout the cytoplasm, while the viral RNA remained associated with vacuolar membranes. Using purified nucleocapsids and ribosomes in vitro, we established that ribosomes alone were sufficient for uncoating. Their role was to release the capsid proteins from nucleocapsids and irreversibly sequester them, in a process independent of energy and translation. The process was stoichiometric rather than catalytic, with a maximum of three to six capsid proteins bound to each ribosome. More than 80%o of the capsid proteins could thus be removed from the viral RNA, resulting in the formation of nucleocapsid remnants whose sedimentation coefficients progressively decreased from 140S to 80S as uncoating proceeded.

“…Digestion with benzonase removed the nucleic acid components without affecting the stability of these capsid assemblies (18). In contrast, while the isolated alphavirus NC is also permeable to RNase digestion, the RNA is needed to maintain NC stability (78,79). Cryo-electron tomography of the RUBV NC-like structures reveals pseudotetrameric arrays of capsid dimers, which are also observed in RUBV particles (18).…”

Section: Structure Of Rubvmentioning

confidence: 99%

Molecular and Structural Insights into the Life Cycle of Rubella Virus

Das

Kielian

2021

Rubella virus (RUBV), a rubivirus, is an airborne human pathogen that generally causes mild measles-like symptoms in children or adults. However, RUBV infection of pregnant women can result in miscarriage or congenital rubella syndrome (CRS), a collection of long-term birth defects including incomplete organ development and mental retardation. Worldwide vaccination campaigns have significantly reduced the number of RUBV infections, but RUBV continues to be a problem in countries with low vaccination coverage. Further, the recent discovery of pathogenic rubiviruses in other mammals emphasizes the spillover potential of rubella-related viruses to humans. In the last decade, our understanding of RUBV has been significantly increased by virological, biochemical, and structural studies, providing a platform to begin understanding the life cycle of RUBV at the molecular level. This review concentrates on recent work on RUBV, focusing on the virion, its structural components, and its entry, fusion, and assembly mechanisms. Important features of RUBV are compared with those of viruses from other families. We also use comparative genomics, manual curation, and protein homology modeling to highlight distinct features of RUBV that are evolutionarily conserved in the non-human rubiviruses. Since rubella-like viruses may potentially have higher pathogenicity and transmissibility to humans, we also propose a framework for utilizing RUBV as a model to study its more pathogenic cousins.