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Measles virus phosphoprotein gene products: conformational flexibility of the P/V protein amino-terminal domain and C protein infectivity factor function.

ABSTRACT: The measles virus (MV) P gene codes for three proteins: P, an essential polymerase cofactor, and V and C, which have multiple functions but are not strictly required for viral propagation in cultured cells. V shares the amino-terminal domain with P but has a zinc-binding carboxyl-terminal domain, whereas C is translated from an overlapping reading frame. During replication, the P protein binds incoming monomeric nucleocapsid (N) proteins with its amino-terminal domain and positions them for assembly into the nascent ribonucleocapsid. The P protein amino-terminal domain is natively unfolded; to probe its conformational flexibility, we fused it to the green fluorescent protein (GFP), thereby also silencing C protein expression. A recombinant virus (MV-GFP/P) expressing hybrid GFP/P and GFP/V proteins in place of standard P and V proteins and not expressing the C protein was rescued and produced normal ratios of mono-, bi-, and tricistronic RNAs, but its replication was slower than that of the parental virus. Thus, the P protein retained nearly intact polymerase cofactor function, even with a large domain added to its amino terminus. Having noted that titers of cell-associated and especially released MV-GFP/P were reduced and knowing that the C protein of the related Sendai virus has particle assembly and infectivity factor functions, we produced an MV-GFP/P derivative expressing C. Intracellular titers of this virus were almost completely restored, and those of released virus were partially restored. Thus, the MV C protein is an infectivity factor.

SUBMITTER: Devaux P

PROVIDER: S-EPMC523285 | biostudies-other | 2004 Nov

REPOSITORIES: biostudies-other

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Project description:Measles virus (MeV), like all viruses of the order Mononegavirales, utilizes a complex consisting of genomic RNA, nucleoprotein, the RNA-dependent RNA polymerase, and a polymerase cofactor, the phosphoprotein (P), for transcription and replication. We previously showed that a recombinant MeV that does not express another viral protein, C, has severe transcription and replication deficiencies, including a steeper transcription gradient than the parental virus and generation of defective interfering RNA. This virus is attenuated in vitro and in vivo However, how the C protein operates and whether it is a component of the replication complex remained unclear. Here, we show that C associates with the ribonucleocapsid and forms a complex that can be purified by immunoprecipitation or ultracentrifugation. In the presence of detergent, the C protein is retained on purified ribonucleocapsids less efficiently than the P protein and the polymerase. The C protein is recruited to the ribonucleocapsid through its interaction with the P protein, as shown by immunofluorescence microscopy of cells expressing different combinations of viral proteins and by split luciferase complementation assays. Forty amino-terminal C protein residues are dispensable for the interaction with P, and the carboxyl-terminal half of P is sufficient for the interaction with C. Thus, the C protein, rather than being an "accessory" protein as qualified in textbooks so far, is a ribonucleocapsid-associated protein that interacts with P, thereby increasing replication accuracy and processivity of the polymerase complex.IMPORTANCE Replication of negative-strand RNA viruses relies on two components: a helical ribonucleocapsid and an RNA-dependent RNA polymerase composed of a catalytic subunit, the L protein, and a cofactor, the P protein. We show that the measles virus (MeV) C protein is an additional component of the replication complex. We provide evidence that the C protein is recruited to the ribonucleocapsid by the P protein and map the interacting segments of both C and P proteins. We conclude that the primary function of MeV C is to improve polymerase processivity and accuracy, rather than uniquely to antagonize the type I interferon response. Since most viruses of the Paramyxoviridae family express C proteins, their primary function may be conserved.

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Measles virus phosphoprotein gene products: conformational flexibility of the P/V protein amino-terminal domain and C protein infectivity factor function.

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