M-X-I Motif of Semliki Forest Virus Capsid Protein Affects Nucleocapsid Assembly

119

129

Deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (TBE) virus. These deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. In the context of the full-length TBE genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residues, corresponding to almost 17% of mature protein C, viable virus was recovered. This virus was strongly attenuated but highly immunogenic in adult mice, revealing capsid protein C as a new and attractive target for the directed attenuation of flaviviruses. Apparently, the larger deletions interfered with the correct assembly of infectious virus particles, and this disturbance of virion assembly is likely to be the molecular basis of attenuation. However, all of the mutants carrying large deletions produced substantial amounts of subviral particles, which as judged from density gradient analyses were identical to recombinant subviral particles as obtained by the expression of the surface proteins prM and E alone. The structural and functional flexibility of protein C revealed in this study and its predicted largely alpha-helical conformation are reminiscent of capsid proteins of other enveloped viruses, such as alphaviruses (N-terminal domain of the capsid protein), retroviruses, and hepadnaviruses and suggest that all of these may belong to a common structural class, which is fundamentally distinct from the classical ␤-barrel structures of many icosahedral viral capsids. The possibility of attenuating flaviviruses by disturbing virus assembly and favoring the production of noninfectious but highly immunogenic subviral particles opens up a promising new avenue for the development of live flavivirus vaccines.Members of the genus Flavivirus, family Flaviviridae, such as yellow fever virus, Japanese encephalitis virus, West Nile virus, the dengue viruses, and tick-borne encephalitis (TBE) virus, cause major human health problems in large areas of the world (4). In spite of a long and successful history of vaccinations against some flavivirus diseases, there is a continuing demand for the development of new flavivirus vaccines. Flaviviruses are positive-stranded RNA viruses. The genome consists of a single approximately 11-kb-long RNA molecule that encodes all of the viral proteins in a single long open reading frame flanked by a rather short 5Ј and a somewhat longer 3Ј noncoding region (24). For many flaviviruses, including TBE virus, infectious cDNA clones have become available during the past two decades (43). This has made it possible to specifically manipulate the genomes of these viruses. Using this technology, a number of approaches to create attenuated flaviviruses that might be used as live virus vaccines have been pursued, including the construction of chimeric flaviviruses (see reference 2 and references cited there...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Capsid Protein C of Tick-Borne Encephalitis Virus Tolerates Large Internal Deletions and Is a Favorable Target for Attenuation of Virulence

Kofler

Heinz

Mandl

2002

119

129

“…The assembly process to generate an icosahedral core structure from 240 copies of the alphavirus CP and the viral genomic RNA has been investigated in vivo by genetic analyses (3,4,15,16,18,19) and in vitro by using an in vitro assembly system (22, 23, 25, 28, 29) The assembly of the nucleocapsid core requires numerous CP-CP and CP-RNA interactions to generate the highly organized icosahedral core structure. An in vitro core assembly system was developed in order to avoid the complexity of in vivo conditions and has identified several requirements but no clear intermediates for core assembly.…”

Section: Discussionmentioning

confidence: 99%

Alphavirus Capsid Protein Helix I Controls a Checkpoint in Nucleocapsid Core Assembly

Hong

Perera

Kühn

2006

The assembly of the alphavirus nucleocapsid core has been investigated using an in vitro assembly system. The C-terminal two-thirds of capsid protein (CP), residues 81 to 264 in Sindbis virus (SINV), have been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly in vitro. Helix I, which is located in the N-terminal dispensable region of the CP, has been proposed to stabilize the core by forming a coiled coil in the CP dimer formed by the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed core-like particles (CLPs) using an in vitro assembly system. The CPs of SINV and Ross River virus (RRV) do not form phenotypically mixed CLPs, but SINV and Western equine encephalitis virus CPs do form mixed cores. In addition, CP dimers do not form between SINV and RRV in these assembly reactions. In contrast, an N-terminal truncated SINV CP (residues 81 to 264) forms phenotypically mixed CLPs when it is assembled with full-length heterologous CPs, suggesting that the region that controls the mixing is present in the N-terminal 80 residues. Furthermore, this result suggests that the dimeric interaction, which was absent between SINV and RRV CPs, can be restored by the removal of the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto helix I by using domain swapping experiments. Thus, discrimination of the CP partner in alphavirus core assembly appears to be dependent on helix I sequence compatibility. These results suggest that helix I provides one of the important interactions during nucleocapsid core formation and may play a regulatory role during the early steps of the assembly process.

“…In Semliki Forest virus, a related alphavirus, deletions of residues 100 to 105, 106 to 113, and 100 to 113 (SINV numbering) cause defects in NC accumulation (2). The mutation of residues corresponding to SINV CP positions 108 and 110 to alanines in Semliki Forest virus CP also results in the inhibition of NC accumulation (30). Since no NCs devoid of nucleic acid have been found and CP is monomeric in the absence of nucleic acid, CP-nucleic acid interactions are considered to be the initial events in NC assembly.…”

mentioning

confidence: 99%

Role of Sindbis Virus Capsid Protein Region II in Nucleocapsid Core Assembly and Encapsidation of Genomic RNA

Warrier

Linger

Golden

et al. 2008

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.Members of the alphavirus genus are distributed throughout the world and cause encephalitis, rashes, and arthritis in humans (31). Chikungunya virus, a member of this genus, recently caused an epidemic on the Indian Ocean island of Reunion (28). The alphaviruses are composed of a Tϭ4 icosahedral nucleocapsid core (NC) that contains the genomic RNA of the virus. Surrounding the NC is a host-derived lipid bilayer. The glycoproteins E1 and E2, responsible for membrane fusion and receptor recognition, respectively, span this lipid membrane (3,16,19,23,41).The capsid protein (CP) of alphaviruses is a multifunctional protein, acting at different stages in the viral life cycle. It has been shown to act as an autoprotease, to recognize the genomic RNA, and to assemble into an ordered protein shell forming the NC. The NC interacts with viral glycoproteins to produce mature virus. Upon entry into a new host, alphaviruses undergo fusion in the low-pH environment of the endosome and release NCs into the cytoplasm, where they disassemble and release the RNA genome. Although there appear to be different functional domains, some of these activities may correspond to overlapping regions of amino acid residues. In the Sindbis virus (SINV) CP, the protease domain and the region that interacts with glycoproteins have been identified but the regions that specify genome RNA recognition and NC assembly have yet to be fully characterized (34).CP is the first structural protein to be translated from subgenomic RNA, and it cotranslationally cleaves itself from the nascent structural polyprotein (1, 20). The CP then binds to genomic RNA, promoting NC formation. Interaction between the NC and glycoproteins occurs on cellular membranes and results in the budding and release of the virus (31). NCs accumulate in the cell, presumably because the rate of NC assembly is greater than the rate of virus budding. An alternative model of virus assembly, in which the glycoproteins organize CP and genomic RNA into NCs at the plasma membrane and the observed NCs in the cytoplasm ar...