RNA complementary to the genome of RNA tumor viruses in virions and virus-producing cells.

Cells producing type C (avain sarcoma virus) or type B (mouse mammary tumor virus) RNA tumor viruses contain small amounts of RNA complementary to the viral genomes. The negative strands are complementary to at least 30 to 45% of the viral genomes and are found as RNA-RNA duplexes in the nucleus and cytoplasm of infected cells and in mature virions.     

Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions.

Sequences required for efficient packaging of human immunodeficiency virus type 1 (HIV-1) genome RNA into virus particles were identified. Deletion of 19 base pairs between the 5' long terminal repeat and the gag gene initiation codon of HIV-1 resulted in a virus markedly attenuated for replication in human T lymphocytes. The mutant virus was characterized by nearly wild-type ability to encode viral proteins and to produce virion particles. The mutant virions exhibited a significant reduction in the content of HIV-1-specific RNA. These results identify an important component of the HIV-1 packaging signal.     

Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions

A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3' and 80 5' nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.     

Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3' coding region are critical for efficient NS vRNA incorporation and that deletion of the 3' segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3' NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3' end) form a structure that is required for efficient influenza A virus vRNA packaging.     

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.     

Rift Valley fever virus M segment: use of recombinant vaccinia viruses to study Phlebovirus gene expression.

Recombinant vaccinia viruses were constructed and used in conjunction with site-specific antisera to study the coding capacity and detailed expression strategy of the M segment of the Phlebovirus Rift Valley fever virus (RVFV). The M segment could be completely and faithfully expressed in recombinant RVFV-vaccinia virus-infected cells, the gene products apparently being correctly processed and modified in the absence of the RVFV L and S genomic segments. The proteins encoded by the RVFV M segment included, in addition to the viral glycoproteins G2 and G1, two previously uncharacterized polypeptides of 78 and 14 kilodaltons (kDa). By manipulation of RVFV sequences present in the recombinant vaccinia viruses and use of specific antibody reagents, it was found that the 78-kDa protein initiated at the first initiation codon of the open reading frame and encompassed the entire preglycoprotein and glycoprotein G2 coding sequences. The 14-kDa protein appeared to begin from the second in-phase ATG and was composed of only the preglycoprotein sequences. Both viral glycoproteins G2 and G1 could be synthesized and correctly processed in the absence of the 78- and 14-kDa proteins, as well as a large portion of the preglycoprotein sequences. However, the hydrophobic amino acid sequence immediately preceding the mature glycoprotein coding sequences was required for authentic glycoprotein production. The M-segment expression strategy involving aspects of translational initiation and protein processing are discussed. The functional roles of the 78- and 14-kDa proteins remain unclear.     

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene

The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.     

Nucleotide sequence of the Aspergillus nidulans mitochondrial gene coding for the small ribosomal subunit RNA: homology to E. coli 16S rRNA.

The complete primary structure of the 1437 bp gene coding for mitochondrial 15S rRNA and its flanking regions was determined by Maxam-Gilbert sequencing of cloned HindIII fragment H3 of A. nidulans mtDNA. The gene product reveals significant homology (59%) to E. coli 16S rRNA, and the potential secondary structures of both rRNA molecules are very similar, except that the hairpin structures 7, 8 and 30 of the Brimacombe 16S rRNA model are deleted, and that two sequences of 8 and 31 nucleotides are inserted in the mitochondrial species.     

Broad-host-range Yersinia phage PY100: genome sequence, proteome analysis of virions, and DNA packaging strategy

PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.     

Roles of the coding and noncoding regions of rift valley Fever virus RNA genome segments in viral RNA packaging

We characterized the RNA elements involved in the packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5'-terminal 25 nucleotides of each RNA segment were equally competent for RNA packaging and carried an RNA packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA packaging.     

Heterogeneity of the genes coding for 5 S RNA in three related strains of the genus Bacillus.

Summary Three related strains of the genus Bacillus, viz. B. licheniformis, B. subtilis and Bacillus Q were all found to contain a minor species of 5 S RNA in an amount indicating that it is transcribed from only one of the multiple 5 S RNA cistrons known to be present in these strains. The major and minor types of 5 S RNA from each of the three strains could be separated from each other by polyacrylamide gel electrophoresis in the presence of urea. The complete nucleotide sequences of the minor B. subtilis and Bacillus Q 5 S RNAs have been determined. Comparison of these sequences to the previously determined sequence of the minor 5 S RNA from B. licheniformis (Raué et al., 1976) showed the three minor types of 5 S RNA to be identical except for the residues at positions 79, 85 and 95. Moreover, seven out of the eight sequence differences between the major and the minor 5 S RNA are the same in all three strains. Thus, the single cistron coding for minor 5 S RNA has been strongly conserved in all three strains, which may indicate a biological significance for the minor 5 S RNA species.     

Overlapping sequences with high homology to functional proteins coexist on complementary strands of DNA in the rumen bacterium Prevotella albensis.

The potential for two complementary fragments of DNA from a clone from the ruminal bacterium Prevotella albensis to encode sequences with homology to at least part of functional proteins is described. One strand contains a sequence with high homology to dnaK, a member of the hsp70 family, and the other strand contains a sequence with some homology to glutamate dehydrogenase genes. Overlapping of these two genes on opposite strands has been reported in eukaryotic species, and is now reported for the first time in a bacterial species. Further investigation of previously described dnaK genes demonstrates that it is more widespread than might be anticipated, with all thirty other dnaK genes investigated also retaining long sequences encoding at least part of a sequence with high homology to a glutamate dehydrogenase gene.     

Sequence of DNA complementary to a small RNA segment of influenza virus A/NT/60/68.

A small RNA segment from the influenza virus strain A/NT/60/68 (H3N2) was converted to cDNA and then to double-stranded DNA using synthetic oligodeoxynucleotide primers. The double-stranded form was cloned into the bacteriophage M1 3mp7. Clones yielding single-strand recombinant templates in opposite orientation were sequenced by the Sanger dideoxynucleotide chain termination technique. The small viral RNA was 422 nucleotides long and the evidence indicated that it was formed by internal deletion of segment 3. It also contained sequences homologous to segment 1.     

Membrane anchors of vesicular stomatitis virus: characterization and incorporation into virions.

Wild-type vesicular stomatitis virus-infected cells contained multiple carboxy-terminal fragments of the envelope glycoprotein G. They migrated in 16% polyacrylamide gels with two dominant apparent molecular weights, 14,000 and 9,000. Both fragments were immunoprecipitated by two antibodies, anti-G(COOH) and anti-G(stem), made against the last 15 amino acids at the carboxy terminus and against the first 22 amino acids of the ectodomain adjacent to the transmembrane region of G, respectively. Pulse-chase experiments in the presence and absence of tunicamycin indicated that the higher-molecular-weight fragment, Gal, was generated first, presumably in the rough endoplasmic reticulum, and then apparently chased into the faster-migrating, stable fragment, Ga2. Exposure of infected cells to radioactive palmitic acid labeled Ga2. Ga2 was detected in purified virions. These results show that a polypeptide approximately 71 amino acids long is transported and incorporated into budding virions. What signals are operative and whether this C-terminal fragment of G protein is transported as a complex with other viral or host cell proteins are presently unknown.     

Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus.

The genome of bluetongue virus, an orbivirus, consists of 10 double-stranded RNAs, each encoding at least one polypeptide. The smallest RNA segment (S10) encodes two minor nonstructural proteins, NS3 and NS3A, the structures and functions of which are not understood. We have expressed these two proteins in mammalian cells by using the T7 cytoplasmic transient expression system. Using a deletion mutant (lacking the first AUG initiation codon), we have demonstrated that the second initiation codon is used to initiate the synthesis of NS3A protein and that the two initiation codons are responsible for the synthesis not only of NS3 and NS3A but also of high-molecular-weight forms of both proteins. These higher-molecular-weight forms (GNS3 and GNS3A) are glycosylated. We have also demonstrated that the carbohydrate chains of GNS3 and GNS3A could be further modified by heterogeneous extension to polylactosaminoglycan forms. The glycosylated and nonglycosylated forms are found in similar intracellular locations in the Golgi complex. In the presence of cycloheximide, NS3 and NS3A immunofluorescence staining was pronounced in the Golgi complex, confirming that NS3 and NS3A are competent for transport to the Golgi apparatus after synthesis. We conclude that S10 gene products are integral membrane glycoproteins.