Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.     

The complete sequence of RNA segment 2 of influenza A/NT/60/68 P1 protein.

A DNA copy of influenza A/NT/60/68 viral RNA segment 2, corresponding to protein P1, has been cloned in the E.coli plasmid pBr322. The clone is 2341 nucleotides long and represents a full-length copy of the viral RNA. In the viral complementary (plus sense) strand there is an open reading frame that is 2271 nucleotides long. The predicted primary gene product is a basic 86,300 dalton protein with a net charge at neutral pH of +23. A 29 amino acid stretch of the protein (coded by nucleotide residues 583-669) is highly basic and contains 7 lysine and 8 arginine residues. Other smaller clusters of basic amino acids are also present in the protein.     

Comparison of the sequences and coding of La Crosse and snowshoe hare bunyavirus S RNA species.

The sequence of the S RNA of La Crosse bunyavirus was deduced from analyses of DNA copies cloned in the Escherichia coli plasmid pBR322. The S RNA is 984 nucleotides in length, has a base ratio of 31.8% U, 27.0% A, 23.2% C, and 18.0% G, and codes for two distinct gene products that are read from overlapping reading frames in the viral complementary strand. The larger gene product (N, 26.5 x 10(3) daltons) contains 235 amino acids, and the smaller gene product (NSS, 10.4 x 10(3) daltons) has 92 amino acids. Comparisons with the published sequences of the related snowshoe hare bunyavirus S RNA and its gene products (Bishop et al., Nucleic Acids Res. 10:3703-3713, 1982) indicate that there are a total of 114 nucleotide differences (6 additions or deletions and 108 substitutions). Also, there are 22 amino acid differences between the N proteins and 12 amino acid differences between the NSS proteins of the two S RNAs.     

Identification of virus-coded nonstructural polypeptides in bunyavirus-infected cells.

Analyses of bunyavirus-infected cell extracts identified at least two virus-induced nonstructural polypeptides. With snowshoe hare (SSH), La Crosse (LAC), and six SSH-LAC reassortant viruses, it was shown that one of these nonstructural polypeptides (NSs, approximate molecular weight, 7.4 X 10(3)) is coded by the SSH small (S)-size viral RNA species. This nonstructural polypeptide was not detected (at least in the same relative abundancies) in LAC virus-infected cells or in cells infected with reassortants having LAC S RNA. For SSH virus, tryptic peptide analyses of either [3H]leucine- or [3H]arginine-labeled NSs indicated that it contains unique sequences not present in the SSH nucleocapsid (N) polypeptide (also coded by the S RNA; J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978). Analyses of SSH virus-infected cell extracts and extracts of cells infected with SSH-LAC reassortants having SSH medium (M)-size RNA species indicated that a nonstructural polypeptide (NSM; approximate molecular weight, 12 X 10(3)) is coded by the SSH M RNA species. In extracts of LAC virus-infected cells (or cells infected with SSH-LAC reassortants having LAC M RNA), a polypeptide with an electrophoretic mobility slightly faster than that of the SSH NSM polypeptide was observed (approximate molecular weight, 11 X 10(3)); it has been designated LAC NSM. The relationships of the NSM polypeptides to the other M RNA-coded polypeptides (G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30;767-770, 1979) have not been determined. Two additional polypeptides present in both LAC- and SSH-infected cell extracts also appear to be virus induced (one with an approximate molecular weight of 10 X 10(3), p10; the other with an approximate molecular weight of 18 X 10(3), p18). Whether these polypeptides are virus coded has not been determined.     

Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus.

We determined the complete nucleotide sequence of the small (S) RNA segment of Uukuniemi virus, the prototype of the Uukuvirus genus within the Bunyaviridae family. The RNA, which is 1,720 nucleotides long, contains two nonoverlapping open reading frames. The 5' end of one strand (complementary to the viral strand) encodes the nonstructural protein NSs (273 residues; molecular weight, 32,019), whereas the 5' end of the viral-sense strand encodes the nucleocapsid protein N (254 residues; molecular weight, 28,508). Thus, the S RNA uses an ambisense coding strategy previously described for the S segment of two phleboviruses and the arenaviruses. The localization of the N protein within the S RNA sequence was confirmed by amino-terminal sequence analysis of all five possible cyanogen bromide fragments obtained from purified N protein. Northern (RNA) blot analyses with strand-specific probes showed that the N and NSs proteins are translated from subgenomic mRNAs about 800 and 850 nucleotides long, respectively. These mRNAs are apparently transcribed from full-length S RNAs of opposite polarities. The two mRNA species were also detected in virus-infected cells. Interestingly, highly purified virions contained full-length S RNA copies of both polarities at a ratio of about 10:1. In contrast, virions contained exclusively negative-strand copies of the M RNA segment. The possible significance of these results for viral infection is discussed. The amino acid sequence of the N protein showed 35 and 32% homology (identity) with the N protein of Punta Toro and sandfly fever Sicilian viruses, two members of the Phlebovirus genus. The NSs proteins were much less related (about 15% identity). In addition, the extreme 5' and 3' ends of the S RNA, which are complementary to each other, also showed a high degree of conservation with the two phleboviruses. These results indicate that the uukuviruses and phleboviruses are evolutionarily related and suggest that the two genera could be merged into a single genus within the Bunyaviridae family.     

Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA

The 3'-terminal ends of both the positive and negative strands of the hepatitis C virus (HCV) RNA, the latter being the replicative intermediate, are most likely the initiation sites for replication by the viral RNA-dependent RNA polymerase, NS5B. The structural features of the very conserved 3' plus [(+)] strand untranslated region [3' (+) UTR] are well established (K. J. Blight and C. M. Rice, J. Virol. 71:7345-7352, 1997). However, little information is available concerning the 3' end of the minus [(-)] strand RNA. In the present work, we used chemical and enzymatic probing to investigate the conformation of that region, which is complementary to the 5' (+) UTR and the first 74 nucleotides of the HCV polyprotein coding sequence. By combining our experimental data with computer predictions, we have derived a secondary-structure model of this region. In our model, the last 220 nucleotides, where initiation of the (+) strand RNA synthesis presumably takes place, fold into five stable stem-loops, forming domain I. Domain I is linked to an overall less stable structure, named domain II, containing the sequences complementary to the pseudoknot of the internal ribosomal entry site in the 5' (+) UTR. Our results show that, even though the (-) strand 3'-terminal region has the antisense sequence of the 5' (+) UTR, it does not fold into its mirror image. Interestingly, comparison of the replication initiation sites on both strands reveals common structural features that may play key functions in the replication process.     

Evidence for a partial RNA transcript of the small circular component of kinetoplast DNA of Crithidia acanthocephali.

The major component of kinetoplast DNA (kDNA) in the protozoan Crithidia acanthocephali is an association of approximately 27,000, 0.8 micrometers (1.58 x 10(6) dalton) circular molecules apparently held together in a particular structural configuration by topological interlocking. We have carried out hybridization experiments between kDNA samples containing one or the other of the two complementary (H and L) strands of purified 0.8 micrometers molecules derived from mechanically disrupted associations and RNA samples prepared either from whole C. acanthocephali cells or from a mitochondrion-enriched fraction. The results of experiments involving cesium sulfate buoyant density centrifugation indicate that whole cell RNA contains a component(s) complementary to all kDNA H strands, but none complementary to kDNA L strands. Similar results were obtained using mitochondrion-associated RNA. Digestion of RNA/DNA hybrids and suitable controls with the single-strand-specific nuclease S1 indicated that 10% of the kDNA H strand is involved in hybrid formation. Visualization of RNA/DNA hybrids stained with bacteriophage T4 gene 32 protein revealed that hybridation involves a single region of each kDNA H strand, equal to approximately 10% of the molecule length. These data suggest that at least 10% of the small circular component of kDNA of Crithidia acanthocephali is transcribed.     

Complete sequence of bluetongue virus L2 RNA that codes for the antigen recognized by neutralizing antibodies.

The complete sequence of the RNA which encodes the major outer-shell-neutralizing antigen (VP2) of bluetongue virus serotype 10 was determined from overlapping cDNA clones inserted into pBR322. The segment L2 RNA was 2,926 base pairs long (1.87 X 10(6) daltons) and had, in one strand, an open reading frame capable of coding for a protein that had a calculated size of 111,122 daltons (956 amino acids) and a +11.5 net charge. The coding strands of both the L2 gene and the group-specific L3 gene of bluetongue virus serotype 17 (M. Purdy, J. Petre, and P. Roy, J. Virol. 51:754-759, 1984) had common sequences of some six nucleotides at their 5' termini (namely, GUUAAA...) and eight nucleotides (namely, ...ACACUUAC) at their 3' termini. Both had short 5' noncoding regions with AUG codons at residues 20 to 22 (L2) and 18 to 20 (L3). The sequences flanking these AUG codons were similar (A/GCCAUGG). The 3' noncoding regions were longer (36 nucleotides for L2, 49 nucleotides for L3). The predicted amino acid sequence of the L2, compared with the similarly sized L3 gene product, was rich in cysteine residues and charged amino acids.     

Transcription In Vitro by Reovirus-Associated Ribonucleic Acid-Dependent Polymerase

Digestion of purified reovirus type 3 with chymotrypsin degrades 70% of the viral protein and converts the virions to subviral particles (SVP). The SVP contain 3 of the 6 viral structural proteins and all 10 double-stranded ribonucleic acid (RNA) genome segments but not adenine-rich, single-stranded RNA. An RNA polymerase which is structurally associated with SVP transcribes one strand of each genome segment by a conservative mechanism in vitro. The single-stranded products include large (1.2 x 10(6) daltons), medium (0.7 x 10(6) daltons), and small (0.4 x 10(6) daltons) molecules which hybridize exclusively with the corresponding genome segments. The enzyme obtained by heating virions at 60 C synthesizes similar products. Kinetic and pulse-chase studies indicate that the different-sized products are synthesized simultaneously but at rates which are in the order: small > medium > large.     

Cell proteins TIA-1 and TIAR interact with the 3' stem-loop of the West Nile virus complementary minus-strand RNA and facilitate virus replication

It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3'-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3'(-) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3'(-) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3'(-) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3'(-) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (K(d)) for the interaction between TIAR RRM2 and the WNV 3'(-) SL RNA was 1.5 x 10(-8), while that for TIA-1 RRM2 was 1.12 x 10(-7). WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.     

Preliminary physical mapping of RNA-RNA linkages in the genomic RNA of Moloney murine leukemia virus

Retrovirus particles contain two copies of their genomic RNA, held together in a dimer by linkages which presumably consist of a limited number of base pairs. In an effort to localize these linkages, we digested deproteinized RNA from Moloney murine leukemia virus (MLV) particles with RNase H in the presence of oligodeoxynucleotides complementary to specific sites in viral RNA. The cleaved RNAs were then characterized by nondenaturing gel electrophoresis. We found that fragments composed of nucleotides 1 to 754 were dimeric, with a linkage as thermostable as that between dimers of intact genomic RNA. In contrast, there was no stable linkage between fragments consisting of nucleotides 755 to 8332. Thus, the most stable linkage between monomers is on the 5' side of nucleotide 754. This conclusion is in agreement with earlier electron microscopic analyses of partially denatured viral RNAs and with our study (C. S. Hibbert, J. Mirro, and A. Rein, J. Virol. 78:10927-10938, 2004) of encapsidated nonviral mRNAs containing inserts of viral sequence. We obtained similar results with RNAs from immature MLV particles, in which the dimeric linkage is different from that in mature particles and has not previously been localized. The 5' and 3' fragments of cleaved RNA are all held together by thermolabile linkages, indicating the presence of tethering interactions between bases 5' and bases 3' of the cleavage site. When RNAs from mature particles were cleaved at nucleotide 1201, we detected tethering interactions spanning the cleavage site which are intramonomeric and are as strong as the most stable linkage between the monomers.     

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.     

Nucleotide sequence of reovirus genome segment S3, encoding non-structural protein sigma NS.

This report describes the complete nucleotide sequence of human reovirus (Dearing strain) genome segment S3. Previous studies indicated that this RNA encodes the major non-structural viral polypeptide sigma NS, a protein that binds ssRNAs (Huisman & Joklik, Virology 70, 411-424, 1976) and has a poly(C)-dependent poly(G) polymerase activity (Gomatos et al., J. Virol. 39, 115-124, 1981). The genome segment consists of 1,198 nucleotides and possesses an open reading frame that extends 366 codons from the first AUG triplet (residues 28-30). There is no significant sequence homology between the plus strand of genome segment S3 and that of genome segment S2 determined previously (Cashdollar et al., PNAS 79, 7644-7648, 1982). However, S3 RNA has significant dyad symmetry and regions that can potentially hybridize (delta G = -26 KCal/mole) with S2 RNA. From the predicted amino acid sequence a possible secondary structure for sigma NS protein was determined. Structural features of reovirus RNA and sigma NS are discussed in relation to their role(s) in viral genome assembly.