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.     

Nucleotide sequence and structure of integrated bovine leukemia virus long terminal repeats

Bovine leukemia virus (BLV) proviruses, harbored by the productively infected fetal lamb kidney (FLK-BLV) cell line, were cloned in bacteriophage lambda L47. The nucleotide sequence of the proviral long terminal repeats (LTR) with flanking cell and virus DNA have been determined. The BLV LTR is 531 bp in length and is bounded by the dinucleotides 5'-TG...CA-3', which are part of a 3-bp inverted repeat. The integrated provirus is flanked by 6-bp direct repeats of cellular DNA. A tRNApro primer binding site is present starting 2 bp downstream of the 5' LTR. In addition to sequencing integrated proviral DNA clones, the nucleotide sequence of a cDNA clone, representing the 3' end of genomic viral RNA, was determined; thus revealing the RNA polyadenylation site and R:U5 boundary within the LTR. Unlike most other retroviruses, a consensus polyadenylation signal, "AATAAA," is not located proximal to the BLV polyadenylation site. The RNA initiation site, defining the U3:R boundary, was located in the BLV LTR by S1 nuclease mapping. This site is approximately 25 bp downstream of an A + T-rich region which probably encompasses a Goldberg-Hogness ("TATAA") box and about 90 bp downstream of a potential "CCAAT" box. The BLV LTR possesses a U3 region of 204 bp, an unusually long R region of 241 bp, and a U5 region of 86 bp.     

Nucleotide sequence of the envelope gene of radiation leukemia virus

The nucleotide sequence and the predicted amino acid sequence of the envelope gene (env) of RadLV/VL3 (T+L+) has been determined. RadLV/VL3 (T+L+) is a highly thymotropic (T+) and leukemogenic (L+) murine recombinant retrovirus continuously produced at high titer by BL/VL3 cells, a line established in culture from a radiation leukemia virus (RadLV)-induced C57BL/Ka mouse thymic lymphoma. The envelope gene is strikingly similar (92% homologous) to that encoded by the ecotropic Akv-MuLV. Among the differences, 134 scattered, although unevenly distributed point mutations (6.4%), two 9-bp deletions as well as a 6-bp insertion were observed (which result in 49 amino acid changes in the env gene of RadLV/VL3 (T+L+)).     

Coordination of cleavage of gag and env gene products of murine leukemia virus: implications regarding the mechanism of processing

Mouse 3T6 cells infected with Murine Leukemia Virus (MuLV) were cloned to yield several sublines producing viruses distinct from one another with respect to the ratio of uncleaved to cleaved gag gene-coded polyprotein, Pr65gag. The virus produced by the cloned sublines also differed in the ratio of the env gene-coded protein, p15E, to its product, p12E. The two ratios, Pr65gag/p30 and p15E/p12E, were found to be highly correlated among the cloned cell lines. Velocity gradient separation of the virions produced by individual sublines, followed by polypeptide analysis, demonstrated that the particles were inhomogeneous with respect to extent of cleavage both of PR65gag and of p15E. The two cleavages were again highly correlated. These data indicate that the gag and env gene product cleavages are not independent events but are tightly coupled.     

Maturation of murine leukemia virus env proteins in the absence of other viral proteins

A mutant of Akv which produces env but no detectable gag or pol products (A. Rein, D. R. Lowy, B. I. Gerwin, S. K. Ruscetti, and R. H. Bassin, J. Virol. 41, 626-634 (1982] was examined for maturation of env gene protein products. In comparison with wild-type Akv, the mutant AK 71 synthesizes gPr85env and produces gp70 and Prp15E in normal amounts and with normal kinetics. Cell surface gp70 was found alike in the mutant and wild type. However, cleavage of Pr15E to p15E did not occur in the mutant. This final cleavage step of AK 71 Prp15E could be made to occur by superinfecting cells containing the mutant with baboon endogenous virus. Thus, unlike earlier steps, this final step in maturation of the env gene product appears to require gag or pol gene products. It is proposed that the virus-encoded protease is required for this last step.     

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).     

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.     

Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus

The entire M RNA segment of the phlebovirus Rift Valley fever virus (RVFV) has been molecularly cloned and the complete nucleotide sequence determined. The RNA is 3884 nucleotides in length, corresponding to a molecular weight of 1.38 X 10(6), having a base composition of 27.3% A, 25.4% G, 27.2% U, and 20.1% C. Sequences present at the 3' and 5' termini of the molecule are largely complementary for some 51 residues and can form a stable duplex structure when the potential secondary structure of the entire molecule is considered. A single major open reading frame, capable of encoding 1206 amino acids (131,845 Da), was found in the viral-complementary sequence ("positive" polarity). Amino-terminal amino acid sequencing of the purified viral glycoproteins G1 and G2 allowed for the positioning of the coding sequences for these polypeptides within this major open reading frame in the following orientation with respect to the genomic M RNA: 3'-G2-G1-5'. From the predicted amino acid composition of the two mature viral glycoproteins, both were found to have a high cysteine content (G2, 6%; G1, 5%). Sequences within the open reading frame capable of encoding up to 23,000 Da of polypeptide were found in addition to those required for the viral glycoproteins. The potential contribution of these sequences to the coding capacity of the M RNA, viral protein processing, and intracellular protein distribution is discussed.     

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.     

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.     

Long terminal repeat of Friend-MCF virus contains the sequence responsible for erythroid leukemia

Friend-MCF virus induces erythroid leukemia when injected into newborn NFS mice whereas Moloney virus induces T-cell lymphoma. To identify the portion of Friend-MCF virus responsible for erythroid leukemia induction four in vitro recombinant viruses were constructed in which env regions or U3 regions of LTR were reciprocally exchanged between Friend-MCF and Moloney viruses. A FrMCF-Mol (LTR) virus whose genome was derived primarily from Friend-MCF virus together with 621 nucleotides of Moloney virus at its 3' end including the U3 region of LTR was a thymic lymphoma-inducing virus. A Mol-FrMCF (LTR) virus with the genome derived primarily from Moloney virus but 596 nucleotides of Friend-MCF virus information at the same region as FrMCF-Mol (LTR) was an erythroid leukemia-inducing virus. A Mol-FrMCF (env) virus whose genome was derived primarily from Moloney virus but which had 2.3 kbp of Friend MCF at the 3' end of the pol gene including most of the env gene with all of gp70 and the N terminal of p15E was a lymphoid leukemia-inducing mink cell focus-inducing virus. FrMCF-Mol (env) virus whose genome was derived primarily from Friend-MCF virus but had 2.7 kbp of Moloney virus at the same region as Mol-FrMCF (env) virus was an erythroid leukemia-inducing ecotropic virus. The Mol-FrMCF (LTR) and Mol-FrMCF (env) viruses induced mixed leukemia of erythroid and lymphoid cells in some mice.     

Complete env gene deletions of three replication-defective strains of Rous sarcoma virus and a model for the origin of their genetic structures

Replication-defective deletion mutants of Rous sarcoma virus (RSV) have been described which transform cells in culture and elaborate envelope (-) defective particles. The env deletions of two clonal variants of the Bryan strain of RSV, RSV(-)3, and RSV(-)16, and of a replication-defective variant of Schmidt-Ruppin RSV (SRN8) were analyzed by fingerprinting oligonucleotides hybridized by a molecularly cloned env DNA probe that spans from near the 3' end of pol to the 3' end of env. It was observed that all three replication-defective RSV strains are essentially complete env deletions but retain the 3' end of pol. Based on a common pol-src junction oligonucleotide that may reflect a homologous sequence repeated at both ends of env in nondefective RSV, the env deletions of RSV(-)3 and 16 appear to be isogenic. The original deletion may have involved recombination between these sequences. The absence of this oligonucleotide in SRN8 indicates that the env deletion of SRN8 has different borders and represents an independent env deletion of nondefective RSV. All three defective RSVs have the genetic structure gag-pol-src. This genetic structure is consistent with the need for a complete gag to make a particle and with the assumption that an independent src gene rather than a gag- or gag-pol-src hybrid gene functions in transformation. It is suggested that a complete pol is not necessary for, but may assist, virus particle formation.     

Amino-terminal amino acid sequence and carboxyl-terminal analysis of Rauscher murine leukemia virus glycoproteins

The two glycoproteins of Rauscher murine leukemia virus, gp70 and gp45, were found to have the following identical NH₂-terminal amino acid sequences: Ala-Ala-Pro-Gly-Ser-Pro-His-Gln-Val-Tyr-X-Ile-Thr-X-Glu-Val-(X - unidentified). The COOH-terminal amino acid of gp70 is tyrosine and that of gp45 is leucine. Computer-analyzed amino acid compositional data indicated that the protein moiety of gp45 is smaller than that of gp70. These results are compatible with the suggestion that gp45 is derived from gp70 by proteolytic cleavage.