Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus: comparison with homologous genes from four strains of human and animal rotaviruses

A full-size cloned cDNA copy of the rotavirus gene encoding the structural neutralization glycoprotein (VP7) of Nebraska calf diarrhea virus (NCDV), a strain recently shown to be effective as a vaccine in children, has been sequenced. Comparison of the deduced amino acid sequence of NCDV (serotype 6) VP7 with that of four other rotavirus strains (human WA serotype 1, human HU-5 serotype 2, simian SA-11 serotype 3, and bovine UK serotype 6) indicates that the degree of amino acid homology among VP7 neutralization proteins of these serotypes ranges from 75 to 86%. Four hydrophilic regions at amino acid residues 174-183, 248-256, 287-294, and 310-317 exhibit significant homology and hence may represent common antigenic determinants, while one hydrophilic area at amino acid residues 83-102 exhibits sufficient divergence to suggest it may be involved in serotype specificity.     

Comparison of human and animal rotavirus strains by gel electrophoresis of viral RNA.

Many rotavirus strains have been detected but few have been grown in vitro and this has hampered the development of serologic tests and antigenic comparison of strains obtained from the same or different host species. Because of this limitation of growth in vitro a different approach for distinguishing rotaviruses was undertaken. The rotavirus genome could be separated into 11 discrete RNA segments by polyacrylamide gel electrophoresis. Differences in RNA migration pattern were observed among human strains as well as between human and animal strains; the number of interspecies differences was greater than the number of intraspecies differences. Three distinct patterns were observed among the eight human rotaviruses obtained from each of four successive annual rotavirus epidemics in the Washington, D.C. area. Each of four animal rotaviruses also had distinct patterns which differed from the human patterns in the mobility of from four to seven RNA segments.     

The nucleotide sequence of the env gene and post-env region of bovine leukemia virus

The env gene of a bovine leukemia virus (BLV) tumor-derived proviral DNA clone has been located by comparison of the translated DNA sequence with amino acid sequence data on purified gp60 and p30^env (A. M. Schultz, T. D. Copeland, and S. Oroszlan (1984)Virology135, 417–427). There is a continuous open reading frame from the N terminus of gp60 for 1446 nucleotides; gp60 is predicted to contain 268 amino acids and p30^env 214. The predicted p30^env shows structural features typical of type C viral transmembrane proteins. It is also clearly related to that of the human T-cell leukemia virus (HTLV), as predicted from the DNA sequence of Seiki et al. (M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida (1983)Proc. Natl. Acad. Sci. USA80, 3618–3622) The two proteins show 36% identities in their amino acid sequence, in an alignment requiring six gaps. More distant relatedness is also seen between BLV p30^env and both murine leukemia virus p15E and Rous sarcoma virus gp36. The gp60s of BLV and HTLV are more distantly related than their p30^envs, but their homology is nonetheless statistically significant. Between the presumptive terminator of the env gene and the beginning of the 3′-long terminal repeat is a region of 1817 base pairs of unknown function. Just as in the HTLV post-envelope sequence, there are at least two reading frames which are open for a significant fraction of this region. In neither the tumor-derived clone nor a clone from a virus-producing cell line, however, is there a continuous open reading frame throughout the region. Comparison of the BLV and HTLV sequences within the post-envelope region revealed a very limited but possibly significant similarity.     

Assembly of a temperature-sensitive mutant of Rauscher murine leukemia virus at the cell surface induced by low temperature and by ligands.

NIH/3T3 mouse fibroblasts, infected with a Rauscher murine leukemia virus temperature-sensitive mutant (ts25) defective in assembly of budding particles at 39°, produce virus at the cell surface when the temperature is shifted rapidly to 0°. Virus buds are not assembled within the first 10 min at 0° and gradually increase in number and degree of development over a 2-hr period. However, release of infectious virus could not be demonstrated at 0° and might, therefore, be an energy-dependent process. Significant budding activity was also induced at the nonpermissive temperature by incubating cells with 0.25% glutaraldehyde or with antiserum to the major virus envelope glycoprotein, gp70. Anti-gp70 serum may induce budding by promoting aggregation of gp70-containing molecular assemblies and, consequently, association of core components in some transmembrane fashion. Induction of virus assembly with glutaraldehyde might occur as a results of nonspecific cross-linking of membrane proteins. These results suggest that procedures commonly used to minimize ligand-induced redistribution of cell surface molecules, i.e., labeling at low temperatures or after mild aldehyde fixation, may not immobilize certain membrane-associated molecules.     

The isolation and preliminary characterization of temperature-sensitive transformation mutants of Moloney sarcoma virus.

Temperature-sensitive (ts) transformation mutants of Moloney murine sarcoma virus were isolated from uv-irradiated viral stocks using a selection and screening procedure based on the ability of MSV-transformed NRK cells to grow in methyl cellulose or agar suspension. Mutants isolated by this procedure formed colonies in agar 1000- to 10,000-fold more efficiently at 34° than at 39°. They also exhibited a transformed morphology and elevated hexose transport levels at 34°, but were phenotypically normal at 39°. Both morphology and hexose transport showed transformed → normal and normal → transformed conversion within 12–48 hr of a temperature shift from 34 to 39° and 39 to 34° respectively. In contrast, ts-transformed cells suspended in agar and incubated at 39° for 24 hr showed a 90% reduction in colony-forming ability when the plates were returned to 34°. Superinfection of nonproducer ts-transformed cells with leukemia virus resulted in the rescue of ts MSV. Rescued supernatants also contained a high proportion (10%) of wt MSV. Repeated cloning of ts mutants, either as virus or cells, did not significantly affect the proportion of ts or wt virus rescued. The ability of ts-transformed cells to express the transformed phenotype at 39° could be restored by wt MSV superinfection, but not by MLV superinfection.     

Complete nucleotide sequence of the mRNA coding for the N protein of vesicular stomatitis virus (New Jersey serotype)

The nucleotide sequence of the mRNA encoding the nucleocapsid protein of the New Jersey serotype (Ogden strain) of vesicular stomatitis virus (VSV) was determined from two overlapping cDNA clones spanning almost entirely the coding region of the mRNA. The 5'-terminal noncoding sequence present in the mRNA but not in the cDNA clones was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1329 nucleotides long (excluding polyadenylic acid) and encodes a protein of 422 amino acids. The nucleotide sequence was compared with the previously determined nucleotide sequence of the nucleocapsid protein of the Indiana serotype. An overall identity of 67.7% was found between the two serotypes. The only place where insertions and/or deletions have occurred during the evolution of the two viral genes from their presumed common ancestor is in the untranslated region. The nonidentical nucleotides are distributed throughout the length of the mRNA although not in an entirely random manner. The predicted amino acid sequence demonstrates that both proteins are initiated from the initiator codon located at the same distance from the 5' end (nucleotides 14 to 16) and contain the same number of amino acids. An overall identity of more than 80% of the amino acid sequence was observed between the two proteins when conservative replacements of amino acids were considered.     

Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins

The complete sequence of the 26 S RNA of Ross River virus (T48 strain) has been obtained and from this the amino acid sequences of the encoded structural proteins have been deduced. These include a basic capsid protein and two envelope glycoproteins. The nucleotide sequence was obtained by chemical sequence analysis of both single-stranded and double-stranded cDNA made to RNA and the sequence data so obtained was rapidly aligned by making use of the protein homology found among the alphaviruses. The polyprotein precursor encoded by the 26 S RNA of Ross River virus is 75% homologous to that of Semliki Forest virus and 48% homologous to that of Sindbis virus. The extent of homology is not uniform within a protein or between proteins and this is discussed with respect to the possible function of the various polypeptide domains in the virus life cycle. In each case the putative attachment site of the amino proximal carbohydrate chains of the three glycoproteins is conserved, whereas the attachment site of a second chain, if present, is not conserved. The 3'-untranslated region of Ross River virus RNA is 524 nucleotides long. It contains a sequence of about 50 nucleotides in length which is present in four copies but which is not shared with other alphaviruses examined.     

Buoyant density in cesium chloride of the human reoviruslike agent of infantile gastroenteritis by ultracentrifugation,electron microscopy,and complement fixation

The buoyant density in cesium chloride of the human reoviruslike (HRVL) agent of infantile gastroenteritis was studied utilizing electron microscopy and complement fixation (CF) for the detection of reoviruslike particles in fractions of the density gradient. Three virus positive stool filtrates were studied. “Full” reoviruslike particles had a density of 1.35–1.37 g/cm³, whereas “empty” particles had a density of 1.29 g/cm³. Peak CF activity coincided with the peak fraction of both the “full” and “empty” reoviruslike particles. In addition, by differential centrifugation, CF activity was also associated with the virion and not a “soluble” antigen.     

Cloning of bovine rotavirus (RF strain): nucleotide sequence of the gene coding for the major capsid protein

The genes of the RF strain of bovine rotavirus have been cloned into pBR 322 following the synthesis and hybridization of cDNA transcribed from both strands of in vitro polyadenylated genomic RNA. Cloned rotavirus DNAs were assigned to most of the 11 genomic RNA segments by Northern blot hybridization. The complete sequence of gene 6 that codes for the major inner capsid protein has been determined. The gene is 1356 nucleotides long and possesses an unique long open reading frame that could encode a protein (397 amino acids) of similar size to the known gene 6 product. Comparison of the RF bovine rotavirus gene 6 sequence with the sequence of the simian rotavirus gene 6, showed these genes to be very similar in nucleotide sequence (87% homology). Most of the base changes are silent and the predicted amino acid sequences are almost identical (97% homology).     

A study of nucleotide sequence homology between strains of tobacco mosaic virus

Nucleotide sequence similarities between the genomes of strains of tobacco mosaic virus have been assessed by a competition-hybridization test. It was found that strains fall into several groups with nucleotide sequences being indistinguishable within a group and without similarity between groups. The strains fall into the same groups whether these are based on capsid protein properties or on nucleotide sequence homology. The two groups represented by strains U1 and dahlemense have capsid proteins which are the most alike of any of the intergroup comparisons, having differences in only 29 or 18% of the 158 sequential amino acid positions and, yet, their nucleic acids show no homology in the competition-hybridization test. Rabbit and mouse globins differ to about the same extent as U1 and dahlemense capsid protein, but in this case, considerable homology has been detected between their genes (Gummerson, R. S., and Williamson, R., Nature (London)297, 265–267, 1974). Possible reasons for the difference in the two systems are discussed.     

The nucleotide sequence of the env gene from the human provirus ERV3 and isolation and characterization of an ERV3-specific cDNA

The nucleotide sequence of the env gene of a previously described human provirus (ERV3) has been determined beginning near the 3'-end of the pol gene and continuing through the 3'-LTR. Analysis of the nucleotide sequence revealed the presence of a long open reading frame of 1944 nucleotides that is capable of encoding a polypeptide that has characteristics of other retroviral glycoproteins and transmembrane proteins. These include the presence of seven potential glycosylation sites, a typical glycoprotein-transmembrane protein cleavage sequence, and amino acid homologies to the glycoproteins and transmembrane proteins of other retroviruses. Further, we have isolated an ERV3-specific cDNA clone from a library prepared from liver RNA of a 20-week human fetus. DNA sequence analysis of this clone revealed that it is identical to the ERV3 genomic clone in the 1110 nucleotides that were sequenced.     

A comparison of the polypeptides of human and bovine respiratory syncytial viruses and murine pneumonia virus

Human and bovine respiratory syncytial (RS) viruses and pneumonia virus of mice (PVM) are morphologically similar viruses and are the only known members of the metamyxovirus (or pneumovirus) group. Comparison of the polypeptides of these viruses by polyacrylamide-gel electrophoresis (PAGE) has shown that the serologically related human and. bovine RS viruses have similar polypeptide profiles. However, PVM does not resemble the other two viruses closely. The molecular weights of RS virus polypeptides were established by discontinuous SDS-PAGE in gradient slab gels and were comparable to previous determinations by continuous SDS-PAGE (Wunner and Pringle, 1976), apart from the detection of an additional 10,000-MW polypeptide. Comparison of 11 strains of human RS virus isolated from different localities, or from the same locality at different dates, showed that the relative mobility of VP32 (a nonglycosylated polypeptide of unknown function) was a stable characteristic of each strain. The mobilities of the other viral polypeptides showed little variation. The 11 strains fell into three groups in which the molecular weights of VP32 were estimated to be 31,000, 32,000, and 35,000, but there was no clear correlation with date or place of origin.     

Nucleotide sequence of a region of the herpes simplex virus type 1 gB glycoprotein gene: mutations affecting rate of virus entry and cell fusion

The tsB5 isolate of herpes simplex virus type I (HSV-1) enters host cells more rapidly than does KOS, an independent isolate of HSV-1, and this rate-of-entry determinant is located between prototypic map coordinates 0.350 and 0.360 (1). The nucleotide sequence of strain tsB5 has now been determined between prototypic map coordinates 0.347 and 0.360. Comparison of the tsB5 sequence to the homologous KOS sequence revealed that the rate-of-entry difference between these two HSV-1 strains may be due to the single amino acid difference observed within these sequences (0.350 to 0.360). A cell fusion determinant in tsB5 is located between coordinates 0.345 and 0.355 and to the left of the rate-of-entry determinant (1). Nucleotide sequence analysis revealed a second amino acid difference between tsB5 and KOS at coordinate 0.349. The cell fusion determinant was tentatively assigned to this location.