Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

Wang, Chun Hsiung; Hsu, Chi Hsin; Wu, Yi Min; Luo, Yu; Tu, Mei Hui; Chang, Wen Ho; Cheng, R. Holland; Lin, Chun‐Fu

doi:10.1007/s11262-010-0488-1

Cited by 16 publications

(8 citation statements)

References 25 publications

(26 reference statements)

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“…The application of different virus purification procedures did not produce direct evidence from virions to reject the result described above. These observations were fully consistent with the previous findings (59), supporting the notion that disulfide bonds participate in virus particle assembly and the stabilization of the capsid structure as reported for other viruses (23,29,33,41,48,52). However, it was surprising that the mutant viruses that completely prevented disulfide-linked N dimerization in virus particles, such as vAN-C23A and vSN-C27A (Fig.…”

Section: Discussionsupporting

confidence: 92%

Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

Zhang

Chen

Sun

et al. 2012

J Virol

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The nucleocapsid (N) proteins of the North American (type II) and European (type I) genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) share only approximately 60% genetic identity, and the functionality of N in both genotypes, especially its role in virion assembly, is still poorly understood. In this study, we demonstrated that the ORF7 3= untranslated region or ORF7 of type I is functional in the type II PRRSV background. Based on these results, we postulated that the cysteine at position 90 (Cys90) of the type II N protein, which corresponds to an alanine in the type I protein, is nonessential for virus infectivity. The replacement of Cys90 with alanine confirmed this hypothesis. We then hypothesized that all of the cysteines in the N protein could be replaced by alanines. Mutational analysis revealed that, in contradiction to previously reported findings, the replacement of all of the cysteines, either singly or in combination, did not impair the growth of either type II or type I PRRSV. Treatment with the alkylating agent N-ethylmaleimide inhibited cysteine-mediated N dimerization in living cells but not in released virions. Additionally, bimolecular fluorescence complementation assays revealed noncovalent interactions in living cells among the N and C termini and between the N-terminal and C-terminal regions of the N proteins of both genotypes of PRRSV. These results demonstrate that the disulfide linkages mediating the N dimerization are not required for PRRSV viability and help to promote our understanding of the mechanism underlying arterivirus particle assembly. P orcine reproductive and respiratory syndrome virus (PRRSV)is an enveloped, single-stranded, positive-sense RNA virus belonging to the family Arteriviridae in the order Nidovirales, which also includes the families Coronaviridae and Roniviridae (7,8,16,47). Other members of the family Arteriviridae include equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) (47). The PRRSV genome is about 15 kb in length, and the coding region is flanked by untranslated regions (UTRs) at each end (the 5= and 3=UTR, respectively) (36). The genome contains at least 10 open reading frames (ORFs), of which ORF1a and ORF1b encode the two long nonstructural polyproteins pp1a and pp1ab and ORF2 to ORF7 encode at least eight structural proteins, glycoprotein 2 (GP2), small envelope (E), GP3, GP4, GP5, ORF5a, membrane (M), and nucleocapsid (N) (13,25,35,36,47,56,61). The structural proteins are expressed from a set of coterminal subgenomic mRNAs (sgmRNAs), which are synthesized by a unique discontinuous transcription strategy (40). The transcription-regulating sequence (TRS) of the leader (5=UTR) and the downstream body TRS (TRS-B) preceding the ORF coding region of each structural protein in the viral genome are believed to play key roles in mediating this process.Based on genetic and antigenic differences, PRRSV strains are classified into two distinct genotypes, North American (t...

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Section: Discussionsupporting

confidence: 92%

Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

Zhang

Chen

Sun

et al. 2012

J Virol

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“…When disulfide bond formation was blocked with NEM, intracellular capsid structures visualized by cryo-EM showed a dramatic loss of specific structural components, including pentons, peripentonal triplexes, and CVSC molecules, as well as a lack of internal DNA and scaffold protein. These results suggest that disulfide bonds contribute to capsid stability, which is reminiscent of other viruses shown to utilize cross-linking to stabilize capsids, including betanodavirus, foot and mouth virus, polyomavirus, retrovirus, and hepatitis B and C viruses (8,17,22,25,33,37,55,61,64,(85)(86)(87).…”

Section: Discussionmentioning

confidence: 88%

Disulfide Bond Formation Contributes to Herpes Simplex Virus Capsid Stability and Retention of Pentons

Szczepaniak

Nellissery

Jadwin

et al. 2011

J Virol

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Disulfide bonds reportedly stabilize the capsids of several viruses, including papillomavirus, polyomavirus, and simian virus 40, and have been detected in herpes simplex virus (HSV) capsids. In this study, we show that in mature HSV-1 virions, capsid proteins VP5, VP23, VP19C, UL17, and UL25 participate in covalent crosslinks, and that these are susceptible to dithiothreitol (DTT). In addition, several tegument proteins were found in high-molecular-weight complexes, including VP22, UL36, and UL37. Cross-linked capsid complexes can be detected in virions isolated in the presence and absence of N-ethylmaleimide (NEM), a chemical that reacts irreversibly with free cysteines to block disulfide formation. Intracellular capsids isolated in the absence of NEM contain disulfide cross-linked species; however, intracellular capsids isolated from cells pretreated with NEM did not. Thus, the free cysteines in intracellular capsids appear to be positioned such that disulfide bond formation can occur readily if they are exposed to an oxidizing environment. These results indicate that disulfide cross-links are normally present in extracellular virions but not in intracellular capsids. Interestingly, intracellular capsids isolated in the presence of NEM are unstable; B and C capsids are converted to a novel form that resembles A capsids, indicating that scaffold and DNA are lost. Furthermore, these capsids also have lost pentons and peripentonal triplexes as visualized by cryoelectron microscopy. These data indicate that capsid stability, and especially the retention of pentons, is regulated by the formation of disulfide bonds in the capsid.Virus capsids have evolved mechanisms to protect viral genomes from the extracellular environment while maintaining the ability to release the viral genome upon entry into a new host cell during the next round of infection. The formation of capsids containing the herpes simplex virus type 1 (HSV-1) double-stranded DNA (dsDNA) genome is a complex process involving a preassembled protein shell (procapsid) containing the viral protease and scaffolding proteins (encoded by UL26 and UL26.5), concatemeric viral DNA, and seven cleavage and packaging proteins (4,13,24). The capsid shell is composed primarily of the major capsid protein (VP5), two triplex proteins (VP19C and VP23), and VP26, as well as a dodecameric UL6 portal ring located at a unique vertex through which DNA most likely is packaged and released.During wild-type (WT) infection, three capsid forms are observed: A capsids, which have participated in an abortive encapsidation process and have lost both scaffold proteins and DNA; B capsids, which contain proteolytically processed forms of the internal scaffold proteins but no DNA; and DNA-containing mature C capsids, which have lost scaffold during the process of taking up DNA (18). DNA-containing C capsids undergo structural alterations which result in the acquisition of increased amounts of a heterodimer made up of UL25 and UL17 (81) believed to stabilize DNA-containing capsids (10,12...

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“…There are four cysteine residues on the GNNV CP (Cys115, Cys187, Cys201 and Cys331). Based on the failure of VLP formation in the presence of single mutations of either C115A or C201A, the existence of a disulfide-bond linkage between Cys115 and Cys201 was previously postulated [ 45 ]. However, a structural inspection of the S-domain of GNNV shows that the distribution of Cys115, Cys187 and Cys201 is too remote to establish intra- or inter-subunit disulfide-bond linkages, implying that the disulfide bond is not required for the proper assembly of the GNNV capsid.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

et al. 2015

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Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.

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Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

Cited by 16 publications

References 25 publications

Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

Disulfide Bond Formation Contributes to Herpes Simplex Virus Capsid Stability and Retention of Pentons

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

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