Short Self-Interacting N-Terminal Region of Rubella Virus Capsid Protein Is Essential for Cooperative Actions of Capsid and Nonstructural p150 Proteins

Abstract

Nucleocapsid formation is a primary function of the rubella virus capsid protein, which also promotes viral RNA synthesis via an unknown mechanism. The present study demonstrates that in infected cells, the capsid protein is associated with the nonstructural p150 protein via the short self-interacting N-terminal region of the capsid protein. Mutational analyses indicated that hydrophobic amino acids in this N-terminal region are essential for its N-terminal self-interaction, which is critical for the capsid-p150 association. An analysis based on a subgenomic replicon system demonstrated that the self-interacting N-terminal region of the capsid protein plays a key role in promoting viral gene expression. Analyses using a virus-like particle (VLP) system also showed that the self-interacting N-terminal region of the capsid protein is not essential for VLP production but is critical for VLP infectivity. These results demonstrate that the close cooperative actions of the capsid protein and p150 require the short self-interacting N-terminal region of the capsid protein during the life cycle of the rubella virus. IMPORTANCEThe capsid protein of rubella virus promotes viral RNA replication via an unknown mechanism. This protein interacts with the nonstructural protein p150, but the importance of this interaction is unclear. In this study, we demonstrate that the short N-terminal region of the capsid protein forms a homo-oligomer that is critical for the capsid-p150 interaction. These interactions are required for the viral-gene-expression-promoting activity of the capsid protein, allowing efficient viral growth. These findings provide information about the mechanisms underlying the regulation of rubella virus RNA replication via the cooperative actions of the capsid protein and p150. R ubella virus (RV) is the sole member of the genus Rubivirus in the family Togaviridae. RV is an enveloped, single-stranded, positive-sense RNA virus with a genome of approximately 10 kb. The genome acts as an mRNA and contains three untranslated regions and two open reading frames (ORFs). The ORF at the 5= end encodes two nonstructural proteins (NSPs), p150 and p90, which function in viral RNA replication. The other ORF, at the 3= end, encodes three structural proteins (SPs), capsid, E2, and E1, which are integral components of the virion (1). NSP p150 contains several domains that are conserved among other RNA viruses (2, 3). A putative methyltransferase domain and a protease domain are located at the amino (N) and carboxyl (C) termini, respectively (2, 4, 5). There are also two domains of unknown function, Y and X, between the methyltransferase and protease domains. The putative methyltransferase domain is considered to play a role in capping viral RNA (4). The protease domain cleaves the precursor polyprotein p200 into p150 and p90, and this step is critical in the regulation of viral RNA replication (6-8). NSP p90 has two functional domains, helicase and RNA-dependent RNA polymerase (RdRp) domains, in the N-and C-terminal reg...

Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins

“…VSV G, P = 0.0003; with anti-VSV G, P = 0.0004; U937 cells without anti-VSV G, P = 0.1670; with anti-VSV G, P = 0.0916; THP-1 cells without anti-VSV G, P = 0.0149; with anti-VSV G, P = 0.0122; Raji cells without anti-VSV G, P = 0.5405 with anti-VSV G, P = 0.1091; M8166 cells without anti-VSV G, P = 0.9232; with anti-VSV G, P = 0.0853; Jurkat cells without anti-VSV G, P = 0.0843; with anti-VSV G, P = 0.0376; MT2 cells without anti-VSV G, P = 0.1080; with anti-VSV G, P < 0.0001. The recombinant rubella virus, rHS, derived from RVi/Hiroshima.JPN/01.03[1J] (Hiroshima) strain, was generated as described previously 33 . The rHS strain was propagated in BHK cells twice to obtain a sufficient stock for the experiments 33 . Antibodies.…”

“…Stock solutions of VSV FLuc -G, VSV FLuc -RV/CE2E1 and RVLP were independently prepared and concentrated and fractionated by sucrose-gradient ultracentrifugation. In this experiment, RVLP, which contains a subgenomic replicon RNA in which the structural genes are replaced with DNA sequences encoding the puromycin N-acetyl-transferase protein, the foot-and-mouth disease virus 2 A self-cleavage domain, and the Renilla luciferase 33 , was used as a marker for RVLP fractionation pattern. VSV FLuc -G, VSV FLuc -RV/CE2E1, and RVLP in individual fractions were detected by infecting Vero cells with the fractions and measuring the relative light unit (RLU) from the cells.…”

“…Plasmid constructs. The expression plasmid encoding the precursor protein for the structural proteins (C, E2 and E1) of the RV Hiroshima strain (pcDNA3.1-SP/C 1-300 ) has been described previously 33 . The expression plasmid encoding only E2 and E1 proteins of the RV Hiroshima strain (pcDNA3.1-E2E1) has been described previously 33 .…”

“…The expression plasmid encoding the precursor protein for the structural proteins (C, E2 and E1) of the RV Hiroshima strain (pcDNA3.1-SP/C 1-300 ) has been described previously 33 . The expression plasmid encoding only E2 and E1 proteins of the RV Hiroshima strain (pcDNA3.1-E2E1) has been described previously 33 . The expression plasmids encoding the envelope proteins of VSV (pC-VSV-G), the Edmonston vaccine strain of MV (pCA7PS-Ed-H and pCXN-Ed-F), and MLV (pFBASALF) have also been described previously 13,[58][59][60] .…”

“…293T cells were transfected with pcDNA3.1-CE2E1 or pcDNA3.1-E2E1 expression plasmids. At 36 h posttransfection, the cells were analyzed by immunoblotting using anti-RV E1 or anti-GAPDH antibody as previously reported 33 . The signal intensity of the E1 protein was normalized by that of GAPDH.…”