The abundant R2 mRNA generated by aleutian mink disease parvovirus is tricistronic, encoding NS2, VP1, and VP2

The abundant R2 mRNA encoded by the single left-end promoter of Aleutian mink disease parvovirus is tricistronic; it not only expresses the capsid proteins VP1 and VP2 but is also the major source for the nonstructural protein NS2. A cis-acting sequence within the NS2 gene was shown to be required for efficient capsid protein production, and its effect displayed a distinct location dependence. Ribosome transit through the upstream NS2 gene region was necessary for efficient VP1 and VP2 expression; however, neither ablation nor improvement of the NS2 initiating AUG had an effect on capsid protein production, suggesting that the translation of the NS2 protein per se had little influence on VP1 and VP2 expression. Thus, proper control of the alternative translation of the tricistronic R2 mRNA, a process critical for viral replication, is governed in a complex manner.     

Efficient transactivation of the minute virus of mice P38 promoter requires upstream binding of NS1.

The P38 promoter of the autonomous parvovirus minute virus of mice is strongly transactivated by the nonstructural protein NS1, a sequence-specific DNA-binding protein. In the context of the complete viral genome, the only unique cis-acting signals required for P38 transactivation by NS1 are the proximal Sp1 site and the TATA element. In the absence of additional upstream sequences, a dependence upon the NS1 binding site within the transactivation response region is observed. Addition of synthetic NS1 binding sites to transactivation response region deletion mutants can restore the ability of NS1 to transactivate P38, and NS1 transactivation has been directly correlated to its ability to bind upstream of the P38 promoter.     

Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies

Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsid proteins (VP5 and VP7), within VIBs in co-transfected or infected cells. Further co-immunoprecipitation analysis confirmed that NS80 could interact with each core protein respectively. In addition, we found that newly synthesized viral RNAs co-localized with VIBs. Furthermore, time-course analysis of viral structural proteins expression showed that the expression of NS80 was detected first, followed by the detection of inner shell protein VP3, and then of other inner-capsid proteins, suggesting that VIBs were essential for the formation of viral core frame or progeny virion. Moreover, knockdown of NS80 by shRNA not only inhibited the expression of aquareovirus structural proteins, but also inhibited viral infection. These results indicated that NS80-based VIBs were formed at earlier stage of infection, and NS80 was able to coordinate the expression of viral structural proteins and viral replication.     

Biochemical characterization of Junonia coenia densovirus nonstructural protein NS-1.

Junonia coenia densovirus (JcDNV) is an autonomous parvovirus that infects the larvae of the common buckeye butterfly, Junonia coenia. Unlike vertebrate parvoviruses, the genes encoding the structural protein and nonstructural (NS) proteins of JcDNV are in opposite orientations; thus, each strand contains a sense and antisense open reading frame (ORF). The promoter at map position 93 controls expression of NS ORFs 2, 3, and 4, which encode three NS proteins, NS-1, NS-2, and NS-3. These proteins are likely to be involved in viral DNA replication, among other functions. In contrast to the nonstructural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been characterized. Here, we describe biochemical properties of the NS-1 protein of JcDNV. The NS-1 ORF was cloned in frame with the Escherichia coli malE gene, which encodes the bacterial maltose binding protein (MBP). Using electrophoretic mobility shift and DNase I protection assays, we identified the region of the JcDNV terminal sequence that is recognized specifically by the MBP-NS-1 fusion protein. The site consists of (GAC)4 and is located on the A-A' region of the terminal palindrome. In addition, the MBP-NS-1 fusion protein catalyzes the cleavage of single-stranded DNA (ssDNA) substrates derived from the JcDNV putative origin of replication, primarily at two sites in the motif 5'-G*TAT*TG-3'. One cleavage site is between the thymidine dinucleotide at positions 92 and 93 and the other site corresponds to thymidine at nucleotide 95; both sites are on the complementary strand of the sequence assigned GenBank accession number A12984. Cleavage of ssDNA is dependent on the presence of a divalent metal cofactor but does not require nucleoside triphosphate hydrolysis. Parvovirus NS proteins contain the phylogenically conserved Walker A- and B-site ATPase motifs. These sites in JcDNV NS-1 diverge from the consensus, yet despite these atypical motifs our analyses support that MBP-NS-1 has ATP-dependent helicase activity. These results indicate that JcDNV NS-1 possesses activities common to the superfamily of rolling-circle replication initiator proteins in general and the parvovirus replication proteins in particular, and they provide a basis for comparative analyses of the structure and function relationships among the parvovirus NS-1 equivalents.     

Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the c-erbA-1 promoter led to the identification of an upstream region that is necessary for NS1-driven transactivation. This sequence harbors a putative hormone-responsive element and is sufficient to render a minimal promoter NS1 inducible in FREJ4 but not in FR3T3 cells, and it is involved in distinct interactions with proteins from the respective cell lines. The NS1-responsive element of the c-erbA-1 promoter bears no homology with sequences that were previously reported to be necessary for NS1 DNA binding and transactivation. Altogether, our data point to a novel, cell-specific mechanism of promoter activation by NS1.     

Localization of the Single-Stranded RNA-Binding Domains of Bluetongue Virus Nonstructural Protein NS2

The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed. Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein. A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations. These truncated proteins were expressed on a large scale and purified to near homogeneity. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays. As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.     

Comparison of promoter activity in Aleutian mink disease parvovirus, minute virus of mice, and canine parvovirus: possible role of weak promoters in the pathogenesis of Aleutian mink disease parvovirus infection.

Aleutian mink disease parvovirus (ADV) infection causes both acute and chronic disease in mink, and we have previously shown that it is the level of viral gene expression that determines the disease pattern. To study the gene regulation of ADV, we have cloned the P3 ADV and P36 ADV promoters in front of a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, and analyzed these constructs by transient transfection in a feline kidney cell line and mouse NIH 3T3 cells. The genes for ADV structural proteins (VP1 and VP2) and the nonstructural proteins (NS-1, NS-2, and NS-3) were cloned into a eukaryotic expression vector, and their functions in regulation of the P3 ADV and P36 ADV promoters were examined in cotransfection experiments. The ADV NS-1 protein was able to transactivate the P36 ADV promoter and, to a lesser degree, the P3 ADV promoter. Constitutive activities of the P3 ADV and P36 ADV promoters were weaker than those of the corresponding promoters from the prototypic parvovirus minute virus of mice (MVM) and canine parvovirus (CPV). Also, the level of transactivation of the P36 ADV promoter was much lower than those of the corresponding P38 MVM and P38 CPV promoters transactivated with MVM NS-1. Moreover, the ADV NS-1 gene product could transactivate the P38 MVM promoter to higher levels than it could transactivate the P36 ADV promoter, while the P36 ADV promoter could be transactivated by MVM NS-1 and ADV NS-1 to similar levels. Taken together, these data indicated that cis-acting sequences in the P36 ADV promoter play a major role in determining the low level of transactivation observed. The P3 ADV and P4 MVM promoters could be transactivated to some degree by their respective NS-1 gene products. However, in contrast to the situation for the late promoters, switching NS-1 proteins between the two viruses was not possible. This finding may indicate a different mechanism of transactivation of the early promoters (P3 ADV and P4 MVM) compared with the late (P36 ADV and P38 MVM) promoters. In summary, the constitutive levels of expression from the ADV promoters are weaker than the levels from the corresponding promoters of MVM and CPV. Moreover, the level of NS-1-mediated transactivation of the late ADV promoter is impaired compared with the level of transactivation of the late promoters of MVM and CPV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of NS3 in cis for virus assembly

We have previously reported successful trans-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol. 74:3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient trans-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for beta-galactosidase (beta-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and beta-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by beta-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in cis for virus assembly.