A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.     

Regulation of RNA synthesis by the genomic termini of vesicular stomatitis virus: identification of distinct sequences essential for transcription but not replication

The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3' leader (Le) and 5' trailer (Tr) regions, were replication competent; however, a 3' copyback replicon (3'CB), containing the natural 3' terminus but having the 5' Tr replaced by a sequence complementary to the 3' Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5' end of 3'CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5' terminus were removed during RNA replication, thus restoring the wild-type 5' Tr to the exact 5' terminus. These data showed that a cis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5' end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5' terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5' terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5' terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5' terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3' copyback defective interfering particles in nature.     

Genome sequences of poa semilatent and lychnis ringspot hordeiviruses

The nucleotide sequences of the genomic RNAs of two hordeiviruses, poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV), were determined. The genome organization of both viruses is similar to that of the type hordeivirus, barley stripe mosaic virus (BSMV). Comparisons of the amino acid sequences of the hordeivirus replicase components revealed a high level of sequence similarity to those of soil-borne wheat mosaic virus (SBWMV), peanut clump virus (PCV), and Indian peanut clump virus. The cysteine-rich gamma b proteins of hordeiviruses are similar to those of SBWMV, PCV, and tobraviruses. Analysis of cis-acting elements in the genomes of the three hordeiviruses revealed conservation of putative core elements of subgenomic RNA promoters. Complex patterns of conservation were also found in the 5'-untranslated regions of hordeivirus genomic RNAs alpha, beta, and gamma.     

Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3'-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5'-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5' leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.     

Prognosis of patients with ovarian cancer and borderline tumours diagnosed in Norway between 1954 and 1993

Double subgenomic Sindbis (dsSIN) viruses were engineered to transduce mosquito cells with antisense RNA derived either from the premembrane (prM) or polymerase (NS5) coding regions of the 17D vaccine strain of yellow fever virus (YFV). Aedes albopictus C6/36 cells were infected at high multiplicities of infection (MOI) with each dsSIN virus. Forty-eight hours later, the transduced cells were challenged with an MOI of 0.1 of the Asibi strain of YFV. At 72-hr postchallenge, the cells were assayed by immunofluorescence for the presence of YFV antigen. Cells transduced with prM or NS5 antisense RNAs derived from the YFV genome displayed no YFV-specific antigens. In contrast, cells infected with control dsSIN viruses that expressed no antisense RNA or dengue virus-derived antisense RNAs were permissive for the challenge virus. To analyze resistance in the mosquito, five log10 50% tissue culture infective doses (TCID50) of each dsSIN virus and three log10TCID50 of either a West African (BA-55) or South American (1899/81) strain of wild-type YFV were coinoculated into Ae. aegypti. Mosquitoes transduced with effector RNAs targeting the prM or NS5 gene regions did not transmit West African YFV and poorly transmitted the South American strain of YFV.     

Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine-adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (-)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3-20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (-)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.