A physical map of caprine arthritis-encephalitis provirus

Terminally redundant linear proviral DNA of approximately 9.5 kb was the major unintegrated species recovered in the Hirt supernatant fraction of caprine synovial membrane cells infected with strain 75-G63 caprine arthritis-encephalitis virus. A physical map based on the cleavage sites of 13 restriction endonucleases was deduced for this proviral DNA.     

Molecular cloning of African swine fever virus DNA

African swine fever virus DNA (about 170 kbp) was cleaved with the restriction endonuclease EcoRI and most of the resulting 31 fragments were cloned in either the phage vector λWES.λB or the plasmid pBR325. Three fragments were not cloned in those vectors, the largest fragment EcoRI-A (21.2 kbp) and the two crosslinked terminal fragments, EcoRI-K′ and D′. Endonuclease SalI cut fragment EcoRI-A into three pieces which were cloned in plasmid pBR322. The two terminal EcoRI fragments were cloned after removal of the crosslinks with nuclease S1 and addition of EcoRI linkers to the fragment ends. The complete library of the cloned fragments accounted for about 98% of ASF virus genome, the missing sequences being those removed by the nuclease S1 in the process of cloning the terminal fragments.     

Molecular cloning of DNA complementary to tobacco vein mottling virus RNA

RNA isolated from tobacco vein mottling virus (TVMV) was used as a template for avian myeloblastosis virus RNA-dependent DNA polymerase, primed with oligo(dT). The largest single-stranded cDNA synthesized was 10 kb, the same as the viral RNA. This material was converted to double-stranded cDNA with Escherichia coli DNA polymerase I and digested with restriction endonuclease HindIII. The cDNA fragments were ligated to HindIII-digested plasmid pBR322 and the product used to transform E. coli strain DG-75. Clones containing recombinant plasmids were selected by ampicillin resistance, and those containing TVMV RNA sequences were selected by colony hybridization with a single-stranded cDNA probe. Four different sizes of recombinant plasmid were reproducibly observed. The inserted DNA portion could be excised in each case with HindIII. The lengths of inserted DNA were 3.0, 1.85, 1.1, and 0.72 kb. A similar procedure was used with PstI-digested cDNA and pBR322. A single type of recombinant plasmid, containing a DNA insertion of 1.85 kb, was reproducibly observed. Hybridization with TVMV RNA confirmed that the five inserted DNA segments were derived from the viral RNA. Hybridization of each recombinant plasmid with the others established that each of the cloned HindIII fragments was unique and that one of them overlapped the cloned PstI fragment. The cloned cDNA fragments were ordered by establishing a restriction map of the cDNA. Together the cloned cDNA fragments account for over 80% of the viral genome.     

Molecular cloning of the complete genome of reovirus serotype 3

All 10 genes of the Dearing strain of reovirus serotype 3 have been cloned in their entirety into pBR322. This has been proved by sequencing the terminal regions (50-100 residues long) of all 10 cloned genes; all such regions were found to be identical to the corresponding terminal regions of the double-stranded RNA genes that are present in reovirus particles. The lengths of the cloned reovirus genes were estimated by comparing their electrophoretic mobilities (and those of their PstI cleavage fragments) with those of accurately known marker DNA molecules. The genes range in size from about 3825 bp for the L genes to about 1200 bp for the S4 gene.     

Complementary DNA cloning and analysis of carnation mottle virus RNA

A 3850-base pair (bp) cDNA fragment, representing most of the carnation mottle virus (CarMV) genome, was molecularly cloned in Escherichia coli using a modification of the plasmid-primer method of Okayama and Berg (H. Okayama and P. Berg (1982), Mol. Cell. Biol. 2, 161-170). Poly(dT)-tailed pUC8 was used to prime cDNA synthesis from polyadenylated CarMV RNA template. Oligo(dC) was added to the cDNA 3' end, and second-strand synthesis was specifically primed using an oligo(dG)-tailed linker fragment. cDNA inserts of 3250 and 1950 by were identified from a clone bank as apparently overlapping and were fused at a common BglII restriction site to produce pCarMV-1C. Confirmation that this plasmid harbored CarMV cDNA sequences was achieved by Southern blot hybridization with a randomly primed cDNA probe. Nick-translated pCarMV-1C was used to probe virion and total single-stranded RNA extracts from infected Chenopodtium quinoa leaves by "Northern" blot hybridization. Besides the full-length genomic RNA, two additional species of approximately 1600 and 1750 bases were associated with CarMV. Based on a series of Northern blots with nick-translated subclones derived from pCarMV-1C as probes, these subgenomic RNAs were found to originate from the 3' domain of the virus genome.     

Molecular cloning and characterization of feline cellular genetic sequences homologous to the oncogene of the McDonough strain of feline sarcoma virus

The organization within the cat genome of cellular genetic sequences homologous to the viral oncogene v-fms of the McDonough strain of feline sarcoma virus (SM-FeSV) was determined. Four cosmid clones containing overlapping v-fms homologous cellular DNA inserts representing a contiguous region of cellular DNA of approximately 80 kbp in length have been isolated from a feline cosmid gene library. Within this region of the cat genome, the c-fms genetic sequences are dispersed over a region of around 30 kbp and are interspersed with at least three intervening sequences.     

Molecular cloning of the PRCII sarcoma viral genome and the chicken proto-oncogene c-fps

The class II avian sarcoma viruses comprise PRCII, PRCIIp, PRCIV, URI, 16L, and Fujinami. The members of this class are all replication-defective viruses containing various amounts of a transforming sequence called v-fps. PRCII contains the smallest amount of fps-specific sequences, transforms fibroblasts in tissue culture, but is only weakly tumorigenic. As a first step in understanding variations in pathogenicity among the class II avian sarcoma viruses and the mechanism by which the oncogene of these viruses was transduced from a single cellular locus, we have molecularly cloned the viral genome of PRCII, its related helper PRCII-AV, and the chicken proto-oncogene (c-fps) from which v-fps derived. The fps-specific region within the cloned PRCII genome was shown to be 0.8-1.0 kb smaller than that of the Fujinami fps-specific region, in agreement with previous studies. Transfection of the cloned DNAs into primary chicken cells demonstrated that both clones (PRCII and PRCII-AV) are biologically active. The cloned PRCII genome is helper dependent and produces a gag-fusion phosphoprotein (P105) which is phosphorylated on a tyrosine residue. The cloned PRCII-AV genome produces infectious virus and can function as a helper for the cloned PRCII genome in transfection assays. Three overlapping recombinant lambda clones homologous to v-fps from a chicken genomic library have been isolated. One of these, lambda-c-fps(2), contains all of the cellular sequences homologous to v-fps. In the aggregate, the three molecular clones may represent the entirety of c-fps.     

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.     

Molecular characterization of two strains of Shope fibroma virus

An isolate of Shope fibroma virus (SFV), designated Indiana (SFV-I), was previously described to be tumorigenic in vivo as SFV, cytocidal in vitro as the orthopoxviruses (vaccinia, rabbitpox, etc.), and to share antigenic determinants with SFV and vaccinia. The genetic relatedness of SFV-I to SFV and vaccinia was studied by means of Southern blotting and hybridization. The results indicated that SFV-I shares extensive DNA homology with vaccinia, but few common sequences with SFV. By contrast, SFV and vaccinia show no sequence homology. These findings suggest that SFV-I is an orthopoxvirus which carries some genetic information from the leporipoxviruses. Recombinants between two genera of poxviruses have not been reported before.     

Characterization, molecular cloning, and physical mapping of the Shope fibroma virus genome

Five strains of Shope fibroma virus (SFV), a strain of rabbit myxoma virus, and a strain of vaccinia virus were compared by restriction endonuclease digestion of their viral DNAs. Restriction digest patterns revealed that SFV and rabbit myxoma, both members of the Leporipoxvirus genus, were distinct from vaccinia, an Orthopoxvirus. All strains of SFV examined had a high degree of nucleotide sequence homology as shown by conservation of restriction sites within their genomes. However, restriction patterns of SFV and myxoma were quite different from one another suggesting that the genomes from these two viruses of the Leporipoxvirus genus do not share a large, highly conserved region of homology as do the viruses belonging to the Orthopoxvirus genus. Restriction mapping identified inverted terminal repeats of approximately 12 kb in length. Restriction fragments representing all but 400 bp of the termini were cloned in plasmid vectors.     

Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith strain)

We have cloned EcoRI and HindIII fragments of the Smith strain of murine cytomegalovirus (MCMV) in the plasmid vector pACYC184. These cloned fragments were used to establish a restriction endonuclease map of the genome with respect to the EcoRI and HindIII sites. The map was constructed on the basis of data derived from cross-hybridizations of EcoRI and HindIII cloned fragments, double-digestions of the cloned fragments with EcoRI and HindIII, and hybridization of cloned HindIII fragments to Southern blots of MCMV DNA cleaved with EcoRI. From our mapping data, we have determined that the length of the MCMV genome is approximately 240 kbp. The genome does not appear to undergo inversions and lacks detectable repeated sequences. One HindIII cloned fragment was obtained which contained both HindIII termini. The existence of this fragment may be related to the mode of replication of the MCMV genome.     

The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectious bursal disease virus

The genome of infectious bursal disease virus (IBDV) strain 002-73 was found to consist of two segments of double-stranded (ds) RNA which were 3400 bp (MW 2.06 X 10(6)) and 2900 bp (MW 1.76 X 10(6)) long, respectively. The ds IBDV RNA could be translated, in vitro, only after extensive denaturation. The small RNA segment was found to code for a single polypeptide of MW 90K, while the large RNA segment coded for three major polypeptides of MW 52K, 32K, and 28K, and two minor polypeptides of MW 41K and 16K. The large RNA segment could encode proteins of MW 125K while the MW of the translated products was 169K suggesting that a precursor-product relationship exists between some of the translation products. A method is described for the synthesis of ds cDNA from large ds RNA molecules. Analyses of recombinant colonies showed that inserts covering the entire IBDV genome had been cloned.     

Molecular cloning and sequencing of the La Crosse virus S RNA

The neutralization of type 1 poliovirus by a monoclonal antibody was studied. The antibody caused polymerization of the virions as observed by sucrose gradient centrifugation and electron microscopy. Dimers, trimers, and higher polymers were formed. No antibody was found in association with the monomeric virions remaining after neutralization, which retained full infectivity. The specific infectivities of dimers, trimers, and higher polymers decreased in that order.     

Use of a focal immunofluorescence assay on live cells for quantitation of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses

A rapid and sensitive focal immunofluorescence assay (FIA) using monoclonal antibodies or heterologous antisera was employed for detection and biological cloning of viruses capable of inducing viral antigens on cell surfaces. The FIA was performed directly on a variety of live cells in tissue culture dishes and was used successfully with C-type murine leukemia viruses (MuLV) of different tropism including ecotropic, xenotropic, amphotropic, and dual-tropic recombinant mink cell focus-inducing (MCF) viruses. With the FIA, we were able to titrate and distinguish ecotropic Friend-MuLV and Friend-MCF viruses present in mixtures. Dual-tropic MCF viruses could be specifically detected directly in mouse cells by using MCF-specific monoclonal antibodies. These antibodies replaced the requirement for production of typical MCF cytopathic effect in mink cells for MCF virus detection, and also allowed efficient titration in mouse cells of MCF virions pseudotyped with ecotropic envelope proteins. Furthermore, by picking foci of fluorescent cells and using their cell-free viral progeny, MCF viruses were cloned from complex pseudotypic mixtures. This allowed the cloning of viruses present at low frequency in heterogeneous mixtures obtained from leukemic tissues.     

Differential effects of defective interfering Semliki Forest virus on cellular and virus polypeptide synthesis

Defective interfering Semliki Forest virus (DI SFV) inhibited virus RNA and virus polypeptide synthesis in cells coinfected with standard virus but did not delay or alter kinetics of RNA synthesis. Inhibition of polypeptide synthesis was 20-fold greater than that of RNA synthesis which presumably reflected the amplification resulting from cumulative translation of mRNAs. At high concentration, DI virus p12e inhibited the shutoff of host protein synthesis and allowed no synthesis of structural or nonstructural polypeptides. Dilution of DI virus restored the inhibition of host protein synthesis but further dilution was necessary before virus-specified polypeptide synthesis could be demonstrated. Another DI virus (p20a) with the same interference titre as p12e also inhibited shutoff of host protein synthesis but synthesis of virus-induced polypeptides was inhibited differentially: significant amounts of polypeptides comigrating with the structural precursor polypeptide p62 and the nonstructural polypeptide nsp63 were present and the synthesis of nsp90 was little affected at any concentration of DI virus p20a tested. None of the DI viruses tested induced the synthesis of any viral or novel polypeptide. It was concluded that DI SFV preparations have qualitatively different interfering activities in relation to their effects on virus and host cell polypeptide synthesis.     

Sites of recombination between the transforming gene of avian myeloblastosis virus and its helper virus

The sites of recombination between the transforming gene of avian myeloblastosis virus (AMV) and its natural helper myeloblastosis-associated virus (MAV) have been determined. In AMV, the cellular sequence substituting for the viral envelope (env) gene gives rise to a different carboxyl terminus of the DNA polymerase. The 5'-recombination site coincides with the RNA splice acceptor site for the production of env mRNA in MAV-infected cells. The 3'-recombination site reveals that the last 11 amino acids including the termination codon are shared by the env protein and AMV transforming protein. The RNA splice acceptor site for the generation of subgenomic v-myb mRNA is located 84 nucleotides downstream from the 5'-recombination site. The AMV transforming protein consists of helper virus-related sequences at both of its amino and carboxyl termini, and all but 84 nucleotides of the cell-derived v-myb sequence. The comparison of MAV gp85 amino acid sequence with those of subgroups B, C, and E indicates that the MAV present in clone lambda 10A2-1 belongs to subgroup B. The high degree of homology among different avian retroviruses of the same subgroup indicates that the amino acid sequence of gp85 is important in determining the conformation of the envelope glycoprotein.