Domain disruptions of individual 3B proteins of foot-and-mouth disease virus do not alter growth in cell culture or virulence in cattle

Picornavirus RNA replication is initiated by a small viral protein primer, 3B (also known as VPg), that is covalently linked to the 5' terminus of the viral genome. In contrast to other picornaviruses that encode a single copy of 3B, foot-and-mouth disease virus (FMDV) encodes three copies of 3B. Viruses containing disrupted native sequence or deletion of one of their three 3B proteins were derived from a FMDV A24 Cruzeiro full-length cDNA infectious clone. Mutant viruses had growth characteristics similar to the parental virus in cells. RNA synthesis and protein cleavage processes were not significantly affected in these mutant viruses. Cattle infected by aerosol exposure with mutant viruses developed clinical disease similar to that caused by the parental A24 Cruzeiro. Therefore, severe domain disruption or deletion of individual 3B proteins in FMDV do not affect the virus' ability to replicate in vitro and cause clinical disease in cattle.     

Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open reading frames (ORFs) 4 and 5 or ORFs 5 and 6 or ORFs 4, 5, and 6 were separated by newly introduced AflII restriction endonuclease cleavage sites. RNA transcribed from each of these plasmids was infectious, demonstrating that the overlapping gene organization is not essential for EAV viability. Moreover, the recombinant viruses replicated with almost the same efficiency, i.e., reached nearly the same infectious titers as the wildtype virus, and stably maintained the mutations that were introduced. The AflII site engineered between ORFs 5 and 6 was subsequently used to generate a virus in which the ectodomain of the ORF 6-encoded M protein was extended with nine amino acids derived from the extreme N-terminus of the homologous protein of mouse hepatitis virus (MHV; family Coronaviridae, order Nidovirales). This nonapeptide contains a functional O-glycosylation signal as well as an epitope recognized by an MHV-specific monoclonal antibody, both of which were expressed by the recombinant virus. Although the hybrid virus had a clear growth disadvantage in comparison to the parental virus, three serial passages did not result in the loss of the foreign genetic material.     

Functional analysis of the grapevine virus A genome

Grapevine virus A (GVA) carries five open reading frames (ORFs). Only the coat protein ORF has been experimentally identified as such; the roles of some of the other ORFs have been deduced by sequence homology to known genes (Minafra et al., 1997). The construction of a full-length, infectious clone of GVA has been previously reported. In an attempt to experimentally define the role of the various genes of GVA, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. Mutations in ORF 1 abolished RNA replication. Mutations in ORF 2 did not affect any of the aforementioned parameters. Mutations in ORFs 3 and 4 restricted viral movement. Mutations in ORF 5 rendered the virus asymptomatic, and partially restricted its movement.     

The complete nucleotide sequence and genome organization of tomato chlorosis virus

Summary. The crinivirus tomato chlorosis virus (ToCV) was discovered initially in diseased tomato and has since been identified as a serious problem for tomato production in many parts of the world, particularly in the United States, Europe and Southeast Asia. The complete nucleotide sequence of ToCV was determined and compared with related crinivirus species. RNA 1 is organized into four open reading frames (ORFs), and encodes proteins involved in replication, based on homology to other viral replication factors. RNA 2 is composed of nine ORFs including genes that encode a HSP70 homolog and two proteins involved in encapsidation of viral RNA, referred to as the coat protein and minor coat protein. Sequence homology between ToCV and other criniviruses varies throughout the viral genome. The minor coat protein (CPm) of ToCV, which forms part of the “rattlesnake tail” of virions and may be involved in determining the unique, broad vector transmissibility of ToCV, is larger than the CPm of lettuce infectious yellows virus (LIYV) by 217 amino acids. Among sequenced criniviruses, considerable variability exists in the size of some viral proteins. Analysis of these differences with respect to biological function may provide insight into the role crinivirus proteins play in virus infection and transmission.     

The genome structure of turnip crinkle virus

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.     

Reverse genetic manipulation of the overlapping coding regions for structural proteins of the type II porcine reproductive and respiratory syndrome virus

The overlapping genomic regions coding for structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) poses problems for molecular dissection of the virus replication process. We constructed five mutant full-length cDNA clones with the overlapping regions unwound and 1 to 3 restriction sites inserted between two adjacent ORFs (ORF1/2, ORF4/5, ORF5/6, ORF 6/7 and ORF7/3′ UTR), which generated the recombinant viruses. Our findings demonstrated that 1) the overlapping structural protein ORFs can be physically separated, and is dispensable for virus viability; 2) such ORF separations did not interrupt the subgenomic RNA synthesis; 3) the plaque morphology, growth kinetics, and antigenicity of these mutant viruses were virtually indistinguishable from those of the parental virus in cultured cells; and 4) these mutant viruses remained genetic stable in vitro. This study lays a foundation for further molecular dissection of PRRSV replication process, and development of genetically tagged vaccines against PRRS.     

Construction of a replicon and an infectious cDNA clone of the Sofjin strain of the Far-Eastern subtype of tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) causes severe encephalitis in humans. The Sofjin-HO strain is the prototype strain of the TBEV Far-Eastern subtype and is highly pathogenic in a mouse model. In this study, we constructed replicons and infectious cDNA clones of the Sofjin-HO strain. The replication of the replicon RNA was confirmed, and infectious viruses were recovered from the infectious cDNA clone. The recombinant viruses showed similar virulence characteristics to those of the parental virus. While characterizing the replicon and infectious cDNA, several amino acid differences derived from cell culture adaptations were analysed. The amino acids differences at E position 496 and NS4A position 58 were found to affect viral replication. The Gly- or Ala-to-Glu substitution at E position 122 was shown to increase neuroinvasiveness in mice. These replicons and infectious cDNA clones are useful in revealing the viral molecular determinants involved in the replication and pathogenicity of TBEV.     

Replication of the RNA segments of a bipartite viral genome is coordinated by a transactivating subgenomic RNA

The insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis. Here we report that RNA1 mutants deficient in RNA3 synthesis failed to support RNA2 replication. This effect was not caused by alterations in the RdRp catalytic subunit nor by a lack of the proteins encoded by RNA3. Furthermore, RNA3 supplied in trans from an exogenous source restored RNA2 replication. These data indicate that RNA3 transactivates the replication of RNA2, a novel property for a viral RNA. We propose that the RNA3 dependence of RNA2 replication serves to coordinate replication of the FHV genome segments.     

Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication

Despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. In the current study, a reverse genetic approach was used to define the role of the 28-kDa amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (MHV) in cell culture. To determine whether nsp1 is required for MHV replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and RNA synthesis. The results demonstrated that the carboxy-terminal half of nsp1 (residues K(124) through L(241)) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. Furthermore, whereas deletion of nsp1 residues amino-terminal to K(124) failed to produce infectious virus, point mutagenesis of the nsp1 amino-terminus allowed recovery of several mutants with altered replication and RNA synthesis. This study identifies nsp1 residues important for protein processing, viral RNA synthesis, and viral replication.     

Sequence of the small double-stranded RNA genomic segment of infectious bursal disease virus and its deduced 90-kDa product

The smaller dsRNA segment of the genome of infectious bursal disease virus (IBDV) encodes a single polypeptide of approximately 90 kDa (VP1). The consensus nucleotide sequence, derived from independent and overlapping cDNA clones, contains a single open reading frame which begins with an exact Kozak sequence and could encode a polypeptide of 878 amino acid residues. It has been suggested that VP1 could be the viral RNA-dependent RNA polymerase. A comparison of the predicted amino acid sequence of this protein with those of other DNA-dependent and ssRNA-dependent RNA polymerases has failed to reveal any homology between VP1 and the conserved regions in these enzymes. It is possible that the polypeptide encoded by the IBDV virus may represent a new class of polymerases which are involved in the replication of double-stranded RNA genomes.     

Nitric oxide radical suppresses replication of wild-type dengue 2 viruses in vitro

Nitric oxide is well accepted as one of the defenses for inhibiting viral dissemination. Macrophages and cells in the macrophage lineage are professional nitric oxide producers which sub-serve as target for dengue virus. The interaction between nitric oxide and dengue virus in such target cell is unknown. In this report, the impact of nitric oxide on infectious dengue virus serotype 2 production and RNA replication was investigated in vitro. Primary isolates of dengue virus serotype 2 from dengue patients were replicated in mouse neuroblastoma cells in the presence of an exogenous nitric oxide donor, s-nitroso-N-acethylpennicillamine, SNAP, at the concentration of 50 or 75 or 100 microM. Nitric oxide inhibited viral replication in a dose and a multiplicity of infection dependent manner. Nitric oxide from 50 and 75 microM SNAP delayed and suppressed replication of dengue virus isolates while higher concentration of nitric oxide, 100 microM SNAP, completely inhibited production of infectious particles up to 36 hr study. Twenty-four out of forty tested isolates, 60%, were susceptible to 50 microM SNAP inhibitory effect. The mechanism of inhibition was investigated at the level of RNA synthesis and was found that RNA production was suppressed which correlated to production of the infectious particles. Down-regulation of the RNA synthesis resulted in reduction of protein synthesis which was detected by lower level of NS1 protein synthesis using immunoblotting. In conclusion, nitric oxide from exogenous nitric oxide donor down regulated replication of dengue virus serotype 2 isolates from dengue patients. The suppression was clearly shown at the level of viral RNA and protein synthesis resulting in reduction of viral progenies production. This phenomenon implies that nitric oxide may serve as a defense which diminishes viral load in patients.     

Microinjected Coxsackie B1 virus does not replicate in HEp-2 cells.

Coxsackie B1 virus did not replicate when microinjected into HEp-2 cells. Replication was assayed by production of infectious virus particles, synthesis of viral RNA, and lysis of cells. The same virus preparation initiated replication when it was inoculated into HEp-2 cells similarly to that by microinjected purified coxsackie B1 virus RNA.     

The M RNA of impatiens necrotic spot Tospovirus (Bunyaviridae) has an ambisense genomic organization.

The nucleotide sequence of Impatiens necrotic spot virus (INSV) M RNA was determined from cDNA clones. The INSV M RNA was 4972 nucleotides in length with two open reading frames (ORFs) in an ambisense genomic organization. The larger ORF near the 3' end of the viral RNA, coding for a protein with a predicted molecular weight of 124.9 kDa, was in the viral complementary sense and produced the G2 and G1 proteins. A smaller ORF in the viral sense was capable of coding for a 34.1-kDa polypeptide, designated the NSm protein. Two subgenomic RNA species were detected in INSV-infected tissue that corresponded to the predicted sizes (3.3 and 1.0 kb) of the G2-G1 and NSm mRNAs. The ORFs were separated by a 478 nucleotide A-U-rich intergenic region similar to the regions found in other viral RNAs with ambisense ORFs. The intergenic region was predicted to form a stable stem-loop structure (-81.2 kcal/mole). The ambisense genomic organization is characteristic of the S RNA for members of the Phlebovirus, Uukuvirus, and Tospovirus genera in the Bunyaviridae family. This is the first report of an ambisense Bunyaviridae M RNA.