Role of the C-terminal tryptophan residue for the structure-function of the alphavirus capsid protein

Skoging, Ulrica; Liljeström, Peter

doi:10.1006/jmbi.1998.1817

Cited by 31 publications

(29 citation statements)

References 40 publications

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“…This encapsidation is dependent on a packaging signal residing in a region of the RNA molecule that codes for the nsP2 protein of the replicase complex (31). Previous studies have shown that wild-type C folds correctly and assembles into NC when expressed as single protein in the absence of spike proteins and in the absence of C self-cleavage from the nascent chain (21,26). Therefore, to investigate whether the mutant capsid proteins assemble into intracellular NCs in the absence of spike proteins, the mutations were subcloned into the SFV-C vector ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Skoging-Nyberg

Liljeström

2001

J Virol

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Alphavirus budding is driven by interactions between spike and nucleocapsid proteins at the plasma membrane. The binding motif, Y-X-L, on the spike protein E2 and the corresponding hydrophobic cavity on the capsid protein were described earlier. The spike-binding cavity has also been suggested to bind an internal hydrophobic motif, M113-X-I115, of the capsid protein. In this study we found that replacement of amino acids M113 and I115 with alanines, as single or double mutations, abolished formation of intracellular nucleocapsids. The mutants could still bud efficiently, but the NCs in the released virions were not stable after removal of the membrane and spike protein layer. In addition to wild-type spherical particles, elongated multicored particles were found at the plasma membrane and released from the host cell. We conclude that the internal capsid motif has a biological function in the viral life cycle, especially in assembly of nucleocapsids. We also provide further evidence that alphaviruses may assemble and bud from the plasma membrane in the absence of preformed nucleocapsids.Semliki Forest virus (SFV) is, together with other alphaviruses, such as Sindbis virus (SINV) and Ross River virus, one of the most extensively characterized enveloped viruses (2, 17, 25). This alphavirus particle has a single positive-strand RNA genome packaged into an icosahedral nucleocapsid (NC) built up from 240 copies of the capsid protein (C) which is arranged in hexameric and pentameric capsomer rings. The NC is surrounded by a lipid bilayer derived from the host cell plasma membrane during budding. A total of 240 envelope protein heterodimers (E1-E2), arranged into 80 spikes, traverse the membrane, and the three E2 tails from one spike complex bind capsid proteins from three separate capsomers. Both the NC and the envelope layer have T ϭ 4 symmetry (2, 7).The structural proteins are translated as polyprotein C-p62 (precursor for E2)-6K-E1 (10). The capsid protein is autoproteolytically cleaved from the nascent chain and remains in the cytoplasm (18). The rest of the polyprotein is translocated into the endoplasmic reticulum via alternating signal and anchor sequences. The proteins are separated by host cell signal peptidases and are transported as a complex to the plasma membrane along the exocytic pathway (11, 15). The current view is that new virus particles are formed at the plasma membrane via interactions between intracellular capsid proteins and the cytoplasmic tails of the E2 spike proteins (26). In this model, an Y-X-L motif in the cytoplasmic domain of E2 interacts with a defined hydrophobic cavity of the capsid protein (19,22,23,32).Determination of a SIN capsid protein crystal structure revealed that the spike-binding cavity harbored an internal hydrophobic peptide, L-X-L, from a neighboring capsid protein molecule, mimicking the docking of the spike Y and L side chains (14). However, in the NC of the mature virion, the capsid monomers are arranged differently than in the crystal, and here the polypeptide with ...

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Section: Resultsmentioning

confidence: 99%

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

Skoging-Nyberg

Liljeström

2001

J Virol

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“…Analogous to NS2, the highly conserved Cterminal tryptophan of the alphavirus capsid occupies the active site postcleavage (4); in fact, distinct similarities have been noted in the catalytic cleft architecture of the two enzymes (21). Mutation of this C-terminal tryptophan to alanine or arginine in a system that uncoupled proteolysis from infectious alphavirus production almost completely abolished nucleocapsid assembly; substitution of the terminal tryptophan with phenylalanine, however, was tolerated (35). Similarly, mutations in the alphavirus capsid that displaced the terminal tryptophan from the active site pocket were found to be highly deleterious to its function (3).…”

Section: Discussionmentioning

confidence: 99%

Determinants of the Hepatitis C Virus Nonstructural Protein 2 Protease Domain Required for Production of Infectious Virus

Dentzer

Lorenz

Evans

et al. 2009

J Virol

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The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.

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“…The coat proteins of the alphavirus organize into spikes and have specializations that aid in maintaining the structure of the virus capsid through interactions with the viral RNA [24]. During virus replication, they are inserted into the lipid bilayer of the host cell to form the viral envelope.…”

Section: Togaviridae Family-genus Alphavirusmentioning

confidence: 99%

“…The other is a highly conserved domain has and has subregions that interact with the cytoplasmic domains. Protein C is responsible for capsid assembly and possesses a serine protease activity, which results in its autocatalytic cleavage and it can also cleave structural proteins [24,51,53].…”

Section: Structure and Function Of The Mayv Proteinsmentioning

confidence: 99%

Mayaro Virus Disease

De-Simone¹

2014

JHVRV

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Mayaro virus produces a nonspecific, sublethal disease in man with symptoms that are often confused with dengue. The symptoms of arthralgia, often associated with these viral infections, can cause an incapacitating disability. To date, outbreaks have been localized and sporadic within the Pan-Amazonia forest since its first isolation in 1954 (Trinidad and Tobago). The available literature is diverse, scarce and dispersed. Mayaro virus is an alphavirus, which is phylogenetically related to the Semliki forest antigenic complex. In the New World, Mayaro and the related UNA viruses are the only members of this complex that have been isolated. The genome of Mayaro consists of single-stranded RNA with a positive charge and a length of 12 kb that can be subdivided into genomic and subgenomic regions that encode nonstructural and structural proteins, respectively. Mayaro has shown great plasticity in hosts for vertebrate infections, but high specificity in invertebrates towards the family Culicidae (mosquitoes). Risk factors for infection are forested areas in northern South America and the rainy season. Two genotypes of MAYV have been identified, L (restricted to Belterra, Brazil) and D (widely distributed in the Pan-Amazonia). The enzootic cycle is similar to the jungle cycle of yellow fever, which involves Haemagogus mosquitoes and monkeys as reservoirs. Of concern is the potential spread of the virus by the involvement of other secondary vectors and hosts such as Aedes aegypti, Aedes albopictus and Aedes scapularis that have been shown experimentally to efficiently transmit the virus. Together with the observed high viremia levels of infected individuals, a significant risk exists for an emerging disease in urban, rural and peridomestic locations close to enzootic foci of Mayaro virus.

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Role of the C-terminal tryptophan residue for the structure-function of the alphavirus capsid protein

Cited by 31 publications

References 40 publications

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

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

Determinants of the Hepatitis C Virus Nonstructural Protein 2 Protease Domain Required for Production of Infectious Virus

Mayaro Virus Disease

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