Genetic assay for multimerization of retroviral gag polyproteins

Luban, Jeremy; Alin, Kimona; Bossolt, K L; Humaran, Teresa; Goff, Stephen P.

doi:10.1128/jvi.66.8.5157-5160.1992

Cited by 64 publications

(26 citation statements)

References 29 publications

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“…Self-interactions of viral nucleocapsid protein (N-N interaction) or multimerization, have been well documented and suggested to be critical to the formation of viral nucleocapsid core, an important composition in the viral particle assembly and maturation in a variety of viral systems [11][12][13][14][15][16][17][18]. The multimerization of nucleocapsid proteins of coronaviruses is possibly a functional form of the protein, which may be involved in the virion assembly by stabilizing the helical structure of the N protein [26].…”

Section: Discussionmentioning

confidence: 99%

“…However, no similar functional roles of SARS-CoV have been reported so far in the literature except that SARS-CoV N may selectively activate AP-1 pathway [10]. Furthermore, while it is of note that self-interaction of nucleocapsid proteins (N-N interaction) and N protein interactions with other viral proteins have been reported to be one of the crucial steps in the formation and assembly of viral particles [11][12][13][14][15][16][17][18]. Such proteinprotein interactions, especially N-N self-interaction in coronaviruses, are poorly documented.…”

mentioning

confidence: 95%

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Analysis of multimerization of the SARS coronavirus nucleocapsid protein

Dobie

Ballantine

et al. 2004

Biochemical and Biophysical Research Communications

170

176

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Severe Acute Respiratory Syndrome (SARS), an emerging disease characterized by atypical pneumonia, has recently been attributed to a novel coronavirus. The genome of SARS Coronavirus (SARS-CoV) has recently been sequenced, and a number of genes identified, including that of the nucleocapsid protein (N). It is noted, however, that the N protein of SARS-CoV (SARS-CoV N) shares little homology with nucleocapsid proteins of other members of the coronavirus family [Science 300 (2003) 1399; Science 300 (2003) 1394]. N proteins of other coronavirus have been reported to be involved in forming the viral core and also in the packaging and transcription of the viral RNA. As data generated from some viral systems other than coronaviruses suggested that viral N-N self-interactions may be necessary for subsequent formation of the nucleocapsid and assembly of the viral particles, we decided to investigate SARS-CoV N-N interaction. By using mammalian two-hybrid system and sucrose gradient fractionations, a homotypic interaction of N, but not M, was detected by the two-hybrid analysis. The mammalian two-hybrid assay revealed an approximately 50-fold increase in SEAP activity (measurement of protein-protein interaction) in N-N interaction compared to that observed in either M-M or mock transfection. Furthermore, mutational analyses characterized that a serine/arginine-rich motif (SSRSSSRSRGNSR) between amino acids 184 and 196 is crucial for N protein oligomerization, since deletion of this region completely abolished the N protein self-multimerization. Finally, the full-length nucleocapsid protein expressed and purified from baculovirus system was found to form different levels of higher order structures as detected by Western blot analysis of the fractionated proteins. Collectively, these results may aid us in elucidating the mechanism pertaining to formation of viral nucleocapsid core, and designing molecular approaches to intervene SARS-CoV replication.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 95%

Analysis of multimerization of the SARS coronavirus nucleocapsid protein

Dobie

Ballantine

et al. 2004

Biochemical and Biophysical Research Communications

170

176

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“…As an additional route to identifying VLP structural elements, we used a yeast two-hybrid system in an attempt to detect subunit interactions. A similar strategy has been employed by Luban et al (1992) and Li et al (1996) with human immunodeficiency virus (HIV) gag and Moloney murine leukaemia virus (MoMLV) env proteins, respectively. Various fragments ( Fig.…”

Section: Determinants Of Protein-protein Interactionmentioning

confidence: 99%

“…3) giving high levels of b -galactosidase activity. As with HIV gag proteins (Luban et al, 1992), it seems therefore that the two-hybrid system is capable of detecting subunit interactions. Deletion of 30 amino acids from the N-terminus of the # 1996 Blackwell Science Ltd, Molecular Microbiology, 22, 667-679…”

Section: Determinants Of Protein-protein Interactionmentioning

confidence: 99%

Structural determinants within the subunit protein of Ty1 virus‐like particles

Martin‐Rendon

Marfany

Wilson

et al. 1996

Molecular Microbiology

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The Ty virus-like particles (VLPs) are functionally analogous to retroviral particles. They package the enzymes and the RNA necessary for retrotransposition, and mediate the integration of the reverse-transcription product into the genome of the host cell. Here we map three structural determinants of particle assembly in the subunit protein. We have also identified key residues in these regions that seem to be involved in subunit interaction and particle morphology. In particular, two point mutations in putative amphipathic helices have remarkable effects on VLP morphology, increasing the diameter as much as eightfold.

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“…Although general functions have been attributed to the mature M-MuLV Gag protein products, little is known of specific protein-protein contacts in immature or mature virions. Some genetic analyses have implicated CA as a major region of intermolecular contacts (17,18,42), and Gag-Gag binding in a yeast two-hybrid system has been attributed to the CA domain (23). The pioneering use of cross-linking agents to examine virus structure (10,32,33) demonstrated that chemical treatment of avian retroviruses produced homotypic dimers of all mature Gag proteins but did not yield any heterotypic dimers (33).…”

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confidence: 99%

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking

Hansen

Barklis

1995

J Virol

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We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimidohexane. Virion-associated wild-type M-MuLV Pr65 Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65 Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65 Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65 Gag protein did not cross-link to the human immunodeficiency virus Pr55 Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65 Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC.

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Genetic assay for multimerization of retroviral gag polyproteins

Cited by 64 publications

References 29 publications

Analysis of multimerization of the SARS coronavirus nucleocapsid protein

Analysis of multimerization of the SARS coronavirus nucleocapsid protein

Structural determinants within the subunit protein of Ty1 virus‐like particles

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking

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