Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro

Abstract

We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was threeto fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.

Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses

“…Despite the attractiveness of hypothesizing that Hsp90 is directly involved in folding viral polymerases, other possibilities exist for how Hsp90 inhibition might destabilize RNA virus polymerases. For viruses of the rhabdovirus and paramyxovirus families, the L polymerase requires the binding of the viral phospho (P) protein for activity (Horikami et al, 1992). Hsp90 may facilitate the L-P interaction to promote the stability of L. Hsp90 could also act to promote the stable formation of L protein multimers (Smallwood, Cevik, and Moyer, 2002).…”

Analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach

“…VSV HR1 is a well characterized mRNA cap methylation defective VSV (Indiana serotype) mutant [26,28,29] with a mutation in the L protein with a D to V substitution at position 1671 [26,27]. This mutation completely eliminates viral mRNA cap methylation at both the guanine-N7 and 2'-O-adenosine positions [26,27] and results in subsequent nontranslatability of primary VSV transcripts in non-permissive cell lines [51][52][53]. As a result, VSV HR1 displays a host-range (hr) phenotype characterized by severely restricted growth in most cell types but only slightly delayed growth in a limited number of "permissive" cells including BHK21 cell line where it achieves wild type titers.…”

Structure and Function of the C-terminal Domain of the Polymerase Cofactor of Rabies Virus

“…[2][3][4][5] The viral RNA-dependent RNA polymerase consists of two subunits, the large protein (L) that contains the polymerase activity and a polymerase cofactor, the phosphoprotein (P) that binds L to the N-RNA. [6][7][8][9][10][11] The polymerase complex cannot transcribe or replicate the naked vRNA; vRNA has to be bound to N in order to be a functional template. 12 P has a second role in the viral life-cycle as a chaperone for N that is not yet bound to vRNA (N 0 ).…”