Identification of a Novel Tripartite Complex Involved in Replication of Vesicular Stomatitis Virus Genome RNA

The genome of the nucleorhabdovirus maize fine streak virus (MFSV) consists of 13,782 nucleotides of nonsegmented, negative-sense, single-stranded RNA. The antigenomic strand consisted of seven open reading frames (ORFs), and transcripts of all ORFs were detected in infected plants. ORF1, ORF6, and ORF7 had significant similarities to the nucleocapsid protein (N), glycoprotein (G), and polymerase (L) genes of other rhabdoviruses, respectively, whereas the ORF2, ORF3, ORF4, and ORF5 proteins had no significant similarities. The N (ORF1), ORF4, and ORF5 proteins localized to nuclei, consistent with the presence of nuclear localization signals (NLSs) in these proteins. ORF5 likely encodes the matrix protein (M), based on its size, the position of its NLS, and the localization of fluorescent protein fusions to the nucleus. ORF2 probably encodes the phosphoprotein (P) because, like the P protein of Sonchus yellow net virus (SYNV), it was spread throughout the cell when expressed alone but was relocalized to a subnuclear locus when coexpressed with the MFSV N protein. Unexpectedly, coexpression of the MFSV N and P proteins, but not the orthologous proteins of SYNV, resulted in accumulations of both proteins in the nucleolus. The N and P protein relocalization was specific to cognate proteins of each virus. The subcellular localizations of the MFSV ORF3 and ORF4 proteins were distinct from that of the SYNV sc4 protein, suggesting different functions. To our knowledge, this is the first comparative study of the cellular localizations of plant rhabdoviral proteins. This study indicated that plant rhabdoviruses are diverse in genome sequence and viral protein interactions.

Section: Discussionmentioning

confidence: 99%

Complete Genome Sequence and In Planta Subcellular Localization of Maize Fine Streak Virus Proteins

Tsai¹,

Redinbaugh

Willie³

et al. 2005

“…Internal transcription initiation has been long suggested for VSV (31,32), and more recently, VSV replication and transcription were shown to be initiated at different positions on the VSV genome (35). Polymerase complexes with different protein contents that are active either in replication or in transcription have been described (17). The existence of individual replicase and transcriptase forms of the polymerase has also been suggested for RV by analyses of gene expression of recombinant ambisense RVs (10) In this study, we have described the first recombinant RVs with clearly distinguishable phenotypes of RNA synthesis regulation caused by a single point mutation in M protein and demonstrated that the role of M in regulation of intracellular RNA synthesis is genetically dissociable from morphogenesis functions.…”

Section: Discussionmentioning

confidence: 99%

Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly

Finke

Conzelmann

2003

Matrix (M) proteins of negative-strand RNA viruses play a crucial role throughout the virus life cycle. The functions of the M proteins in virus assembly and morphogenesis and in budding off enveloped virus particles have been extensively studied and well recognized. More recently, reverse genetics experiments with rabies virus (RV), a member of the Rhabdoviridae family, revealed an additional and unexpected function of rhabdovirus M proteins apart from virus assembly, namely, the regulation of RNA synthesis and profoundly affecting the balance of replication and transcription products (13).The viral RNA of nonsegmented negative-strand RNA viruses (order Mononegavirales) is associated with the viral nucleoprotein (N) to form a tight ribonucleoprotein (RNP) complex. Such RNPs are the template for transcription of subgenomic virus mRNAs and for replication of full-length virus RNAs. The latter probably involves concurrent encapsidation of the nascent RNA into RNPs. A constant supply of N protein is therefore a prerequisite for productive replication (1, 28). In contrast, transcription from the RNP does not need a supply of N. Both RNA replication and transcription are performed by the virus-encoded RNP-dependent RNA polymerase, which is composed of a large, catalytically active subunit (L) and a polymerase cofactor, the phosphoprotein P. P also has an important role during replication, as N-P complexes, rather than N alone, are required for proper and selective encapsidation of viral RNA (8,20). After infection, replication is therefore not possible until the so-called primary transcription and translation provide enough N protein.In spite of the fact that N is the most abundant viral protein in infected cells at later stages, transcription is the predominant RNA synthesis mode throughout infection, arguing against a simple and popular model in which the amount of available N protein or N-P complexes would determine the level of replication versus transcription. This is supported by reverse genetics experiments with RV (unpublished), vesicular stomatitis virus (VSV) (34), and respiratory syncytial virus (RSV) (9). The ratio of replication and transcription was found to be fixed and could not be altered by providing variable levels of N protein. Even with limiting amounts of N protein, only the overall RNA synthesis was reduced, suggesting that other trans-acting factors are responsible for balancing replication and transcription. Indeed, the RSV M2-2 protein, which is unique to members of the Pneumovirus genus and is associated with RNPs, was shown to be a trans-acting factor able to shift the balance of RNA synthesis from transcription toward RNA replication (2).Reverse genetics experiments with RV minigenomes, Mgene deletion mutants, and recombinant full-length virus with modified M expression more recently revealed an important function of the M protein in the regulation of rhabdovirus RNA synthesis. In the absence of M, replication of RV was downregulated, while the transcription efficiency was enhanced, resul...

“…One possibility is that nt 16 to 34 encode an encapsidation nucleation site but that encapsidation is mediated by the polymerase during RNA synthesis. Alternatively, it is possible that these nucleotides constitute a signal in the genome template, rather than nascent antigenome, that is important for recruiting a polymerase that is able to mediate encapsidation, for example, a polymerase that is preloaded with N protein, which has been suggested to be the active form of the VSV replicase (18,31).…”

Section: Discussionmentioning

confidence: 99%

Identification of Internal Sequences in the 3′ Leader Region of Human Respiratory Syncytial Virus That Enhance Transcription and Confer Replication Processivity

McGivern

Collins

Fearns

2005

Previous studies of respiratory syncytial virus have shown that the 44-nucleotide (nt) leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nt at the 3 end of Le. In the present study the remainder of the Le region was mapped to identify sequences important for transcription and replication. A series of minigenomes with mutant Le sequences was generated, and their ability to direct transcription and replication was determined by Northern blot analysis, which examined full-length antigenome and mRNA, and by primer extension analysis, which examined antigenome and mRNA initiation. With regard to transcription, nt 36 to 43, located immediately upstream of the GS signal, were found to be necessary for optimal levels of mRNA synthesis, although the GS signal in conjunction with the 3-terminal region of Le was sufficient to direct accurate mRNA synthesis initiation. With regard to replication, the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome. Analysis of transcripts produced from di-and tricistronic minigenomes indicated that a significant proportion of abortive replicases continue RNA synthesis to the end of the first gene and then continue in a transcription mode along the remainder of the genome.