bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA
Authors: DiMaio D
Published: January 2020

Oligonucleotide-directed mutagenesis was used to construct a nonsense mutation in open reading frame (ORF) E2 of bovine papillomavirus DNA. A single base substitution mutation was constructed which converted a TAC codon into a TAG amber stop codon at a position in the ORF that did not overlap with any other viral ORFs. Full-length viral DNA containing the mutation induced only approximately 2% of the transformed foci of mouse C127 cells that were induced by wild-type DNA. In a different transformation assay, approximately one-half of the C127 cells which had acquired the mutant DNA gave rise to colonies containing at least some cells with transformed morphology. The constructed mutation was maintained in cell lines derived from cells which had acquired the mutant viral DNA, but the viral DNA appeared to be integrated into the host cell genome. Genetic mapping experiments proved that the constructed amber mutation caused the decrease in focus-forming activity and the integration of the mutant viral DNA. These results suggest that ORF E2 encodes a protein which is involved either directly or indirectly in some aspects of oncogenic transformation by bovine papillomavirus and in maintaining the viral DNA as a plasmid in transformed cells.
Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing
Authors: La Monica N , Meriam C , Racaniello V R
Published: January 2020

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.
Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A
Authors: Cole C N , Tornow J , Clark R , Tjian R
Published: January 2020

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains.
2.2-kilobase class of early transcripts encoded by cell-related sequences in human cytomegalovirus strain AD169
Authors: Staprans S I , Spector D H
Published: January 2020

In this report we describe the kinetics of appearance and fine mapping of a 2.2-kilobase (kb) class of transcripts arising from a region of the human cytomegalovirus genome which contains cell-related sequences. These transcripts are encoded by adjacent EcoRI fragments R and d (map units 0.682 to 0.713), located within the long unique segment of the genome. The 2.2-kb RNAs were first detected at 8h postinfection and appeared at comparable or slightly lower levels at 28 and 72 h postinfection. At late times (72 h) additional transcripts were detected with probes from this region. RNase, S1 nuclease, and exonuclease VII protection analyses of 8- and 28-h RNA indicated that the 2.2-kb RNAs had a complex spliced structure consisting of invariable 5' and internal exons and a heterogeneous 3' exon. The position of the 5' end of the RNA was determined with respect to the nucleotide sequence. Analysis of this sequence showed that the cell-related sequences were contained within a long open reading frame in the 5' exon.
Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C
Authors: Pincus S E , Diamond D C , Emini E A , Wimmer E
Published: January 2020

Sequence analysis of the genomic RNA of interstrain guanidine-resistant and antibody-resistant variant recombinants of poliovirus type 1 mapped the resistance of mutants capable of growth in 2.0 mM guanidine hydrochloride to a region located 3' of nucleotide 4444. This region of the viral genome specifies the nonstructural protein 2C. The sequence of genomic RNA encoding 2C from six independently isolated mutants resistant to 2.0 mM guanidine was determined. All six isolates contained a mutation in 2C at the same position in all cases, resulting in two types of amino acid changes. Dependent mutants were examined and found to contain two amino acid changes each within 2C. Mutants resistant to 0.53 mM guanidine were isolated and found to lack the mutations seen in variants resistant to 2.0 mM guanidine. A comparison of the amino acid sequences of the 2C proteins of poliovirus, foot-and-mouth disease virus, rhinovirus types 2 and 14, and encephalomyocarditis virus revealed a strong homology over regions totaling 115 residues. All of the mutations observed in guanidine-selected mutants were contained within this region. The amino acid region containing the mutations observed in poliovirus mutants resistant to 2.0 mM guanidine was compared with the homologous region in the other picornaviruses; a strong correlation was found between the amino acid present at this position and the sensitivity of the virus to 2.0 mM guanidine.
T-cell-deficient mice display normal recovery from experimental rotavirus infection
Authors: Eiden J , Lederman H M , Vonderfecht S , Yolken R
Published: January 2020

Rotaviruses are common causes of diarrhea in animals and humans. Little is known, however, about the components of the host response to these viruses. Rotavirus infection was studied in athymic mice experimentally infected with murine rotavirus. Neonatal T-cell-deficient mice experienced a self-limited gastrointestinal infection which was identical to that observed in age-matched immunocompetent mice. Adult T-cell-deficient seronegative mice and age-matched normal mice showed a similar extent of resistance to symptomatic rotavirus infection. In both cases, the infection was resolved without the generation of antirotavirus antibody. These studies indicate that host defense against murine rotavirus requires neither functional T-lymphocytes nor specific antiviral antibody.
Characterization of the genes encoding herpes simplex virus type 1 and type 2 alkaline exonucleases and overlapping proteins
Authors: Draper K G , Devi-Rao G , Costa R H , Blair E D , Thompson R L , Wagner E K
Published: January 2020

A detailed sequence analysis of the herpes simplex virus type 1 (HSV-1) and HSV-2 DNA encoding the alkaline exonuclease mRNA clusters has been completed. Three partially colinear mRNAs (2.3, 1.9, and 0.9 kilobases) are completely encoded within the DNA sequence presented. The putative promoter regions of the transcripts were inserted upstream of a plasmid-borne chloramphenicol acetyl transferase (CAT) gene and assayed for their ability to induce transcription of the CAT gene upon low multiplicity of infection with HSV in transient expression assays. We conclude that the expression of all three transcripts appear to be controlled by individual promoters. The 2.3-kilobase mRNA contains an open translational reading frame sufficient to encode 626 amino acids for the HSV-1 alkaline exonuclease enzyme; this value is 620 amino acids for HSV-2. A comparison of the predicted amino acid sequences of the HSV-1 and HSV-2 alkaline exonuclease enzymes revealed significant amino acid differences in the N-terminal portions of the two proteins; however, computer analyses suggest that the three-dimensional structures of the HSV-1 and HSV-2 nuclease enzymes are very similar. The 0.9-kilobase mRNA contains an open reading frame which shares a small amount of out-of-phase overlap with the C-terminal portion of the alkaline nuclease open reading frame. This open reading frame has the capacity to encode a 96-amino-acid polypeptide (10,500 daltons).
Rous-associated virus 1-induced erythroleukemic cells exhibit a weakly transformed phenotype in vitro and release c-erbB-containing retroviruses unable to transform fibroblasts
Authors: Beug H , Hayman M J , Raines M B , Kung H J , Vennström B
Published: January 2020

Avian leukosis viruses induce erythroblastosis in chicks by integrating into the c-erbB gene and thus activating c-erbB transcription. We characterized Rous-associated virus 1-induced leukemic erythroblasts in vitro and showed that they mostly resemble erythropoietin-independent but otherwise normal erythroid progenitors. Some leukemic cells, however, were able to both differentiate and proliferate extensively in vitro. All 14 leukemias studied expressed high levels of erbB-related proteins that were 5 to 10 kilodaltons larger but otherwise very similar to the gp74erbB protein of avian erythroblastosis virus ES4 with respect to biosynthesis, glycosylation, and cell surface expression. Two leukemias contained and released retroviruses that transduced erbB. Chicken embryo fibroblasts fully infected with these viruses expressed high levels of erbB RNA and protein but retained a normal phenotype. Our results suggest that certain forms of c-erbB, activated by long terminal repeat insertion or viral transduction, are capable of inducing erythroleukemia but unable to transform fibroblasts.
The minute virus of mice P39 transcription unit can encode both capsid proteins
Authors: Labieniec-Pintel L , Pintel D
Published: January 2020

The right-hand 80% of the genome of minute virus of mice (MVM) was cloned into the bovine papillomavirus type I shuttle vector and used to transfect mouse C127 cells. Transformed lines were isolated that efficiently produce both authentic MVM capsid proteins at a ratio similar to that seen in a normal viral infection, and these proteins assemble into intact empty virions. The only transcription of MVM sequences detected in these lines was representative of the viral P39 transcription unit, which therefore contains sufficient information to encode both authentic capsid proteins at the same regulated ratio seen in an infected cell.
Characterization of a myc-containing retrovirus generated by propagation of an MH2 viral subgenomic RNA
Authors: Martin P , Henry C , Ferre F , Bechade C , Begue A , Calothy C , Debuire B , Stehelin D , Saule S
Published: January 2020

We have previously isolated, from wild-type MH2 virus that contains the two oncogenes mil and myc, mutants defective in one or the other oncogene product. We report here the molecular cloning and extensive characterization of MH2 CL25 provirus lacking the v-mil oncogene. Our results indicate that this virus corresponds to the propagation of the 2.8-kilobase subgenomic RNA of MH21.
Neonatal respiratory syncytial virus infection: role of transplacentally and breast milk-acquired antibodies
Authors: Wong D T , Ogra P L
Published: January 2020

The effect of transplacentally and breast milk-acquired antibodies on respiratory syncytial virus infection was studied in neonatal and 2-month-old cotton rats. Adult female rats infected intranasally with live virus regularly produced virus-specific antibodies in the serum, colostrum, and breast milk. By using foster feeding techniques, we showed that both transplacentally and breast milk-acquired antibodies were effective in reducing the replication of respiratory syncytial virus in the lungs of neonatal animals when they were challenged with live virus via the nasal route at 3 days of age. However, the protection provided by these antibodies was rather brief. There was no difference in the replication of respiratory syncytial virus in the lungs of 2-month-old animals that were delivered and nursed by seropositive (immunized) or seronegative (control) cotton rats.
Bovine leukemia virus protease: purification, chemical analysis, and in vitro processing of gag precursor polyproteins
Authors: Yoshinaka Y , Katoh I , Copeland T D , Smythers G W , Oroszlan S
Published: January 2020

Bovine leukemia virus protease was purified to homogeneity and assayed by using murine leukemia virus Pr65gag, a polyprotein precursor of the viral core structural proteins, as the substrate. A chemical analysis of the protease, including an amino acid composition and NH2- and COOH-terminal amino acid sequence analysis, revealed that it has an Mr of 14,000 and is encoded by a segment of the viral RNA located between the gag gene and the putative reverse transcriptase gene. As expected from the nucleotide sequence data (Rice et al., Virology 142:357-377, 1985), the reading frame for the protease is different from both the gag and reverse transcriptase reading frames. The 5' end of the protease open reading frame extends 38 codons upstream from the codon for the NH2-terminal residue of the mature viral protease and overlaps the gag open reading frame by 7 codons. The 3' end of the protease open reading frame extends 26 codons beyond the codon for the COOH-terminal residue of the mature protease and overlaps 8 codons of the reverse transcriptase open reading frame. Several lines of evidence, such as protein mapping of the gag polyprotein precursor, the characteristic structure of the mRNA, and promotion of the synthesis of a gag polyprotein precursor by lysine tRNA in vitro, suggest that the protease could be translated by frameshift suppression of the gag termination codon. In vitro synthesized bovine leukemia virus gag-related polyproteins were cleaved by the protease into fragments which were the same size as the known components of bovine leukemia virus, suggesting that the specificity of cleavage catalyzed in vitro by the purified protease is the same as the specificity of cleavage found in the virus.
RNA virus genomes hybridize to cellular rRNAs and to each other
Authors: McClure M A , Perrault J
Published: January 2020

In this communication we show that the RNA genomes of vesicular stomatitis, Sindbis, and reoviruses can specifically hybridize under stringent conditions to the large rRNAs present in HeLa cell cytoplasmic extracts. In addition, we show that some virus genome RNAs can also hybridize to each other. On the basis of our previous detailed studies identifying specific regions of hybridization between the poliovirus genome and 28S rRNA, we suggest that a similar phenomenon of "patchy complementary" may be responsible for the interactions described here (M. A. McClure and J. Perrault, Nucleic Acids Res. 13:6797-6816, 1985). The possible biological implications of these cross-reacting hybridizations and practical considerations in the use of viral probes for diagnosis are discussed.
Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome
Authors: Fultz P N , McClure H M , Swenson R B , McGrath C R , Brodie A , Getchell J P , Jensen F C , Anderson D C , Broderson J R , Francis D P
Published: January 2020

The lymphadenopathy-associated virus (LAV) prototype strain of human T-lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow-up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact.
A subclass of polyomavirus middle tumor antigen binds to DNA cellulose
Authors: Bolen J B , Cary K , Scheller A , Basilico C , Israel M A , Prives C
Published: January 2020

We examined the binding of polyomavirus large (L-T)-, middle (M-T)-, and small-tumor antigens to DNA cellulose. At pH 6.0, the majority of L-T bound to calf thymus DNA cellulose, while little or no small tumor antigen was retained under these conditions. Unexpectedly, a small but reproducible proportion of M-T bound to both native and denatured DNA cellulose. M-T encoded by polyomavirus mutant dl 8, which expressed shortened L-T and M-T, bound to DNA, indicating that the deleted sequences are not required for DNA binding. Also, M-T from transformed BMT-1 rat cells, which synthesize exclusively this polyomavirus tumor antigen, bound to DNA, indicating that its binding is not due to association with other polyomavirus-encoded proteins. Using the DNA fragment immunoassay, we found that, under conditions in which L-T bound specifically to DNA fragments containing viral regulatory sequences, no viral DNA fragments were bound by M-T. The existence of distinct subpopulations of M-T that differ in their DNA-binding properties was indicated by rebinding experiments in which M-T that had bound to DNA cellulose rebound very efficiently, while that which had not been originally retained by DNA cellulose rebound poorly. Furthermore, the M-T-pp60 c-src complex did not bind to DNA cellulose. These data suggest that polyomavirus M-T is heterogeneous, consisting of populations of molecules that differ in their interactions with DNA cellulose.
Mapping 5' termini of JC virus late RNA
Authors: Kenney S , Natarajan V , Salzman N P
Published: January 2020

The 5' termini of late mRNAs were mapped 17 to 19 days after primary human fetal glial cells were infected with JC virus. The major 5' start sites spanned a region of approximately 250 nucleotides, starting at nucleotide 5114, which was on the early side of the replication origin, and extending to nucleotide 242, which was on the late side of the 98-base-pair (bp) repeats. The sequence TATATAT was contained within each of the 98-bp repeats but does not specify 5' start sites in vivo. However, the sequence TACCTA, which occurred 25 to 30 bp upstream of the simian virus 40 nucleotide position 325 start site (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:625-633, 1982) and functions as a surrogate TATA box, was present 30 bp upstream of two JC virus start sites.
Palindromic structure and polypeptide expression of 36 kilobase pairs of heterogeneous Epstein-Barr virus (P3HR-1) DNA
Authors: Jenson H B , Rabson M S , Miller G
Published: January 2020

Among the Epstein-Barr virions (EBV) produced by the P3HR-1 (HR-1) cell line are a defective subpopulation with rearranged viral DNA designated heterogeneous DNA (het DNA). These defective virions are responsible for the capacity of HR-1 virus to induce early antigen in Raji c cells and for trans activation of latent EBV in X50-7 cells. Virions with het DNA are independent replicons which pass horizontally from cell to cell rather than being partitioned vertically. We analyzed the structure and defined several polypeptide products of het DNA to understand these remarkable biologic properties. A 36-kilobase-pair (kbp) stretch of het DNA was cloned (as two EcoRI fragments of 20 and 16 kbp) from virions released from a cellular subclone of HR-1 cells. The unusual aspect of the 20-kbp fragment was the linkage of sequences of BamHI-M and BamHI-B', which are not adjacent on the standard EBV genome. The 16-kbp fragment was a palindrome in which at least two additional recombinations on each side of the palindrome had linked regions of the standard EBV genome which are not normally contiguous. The 20-kbp het DNA fragment was attached to at least one and possibly both ends of the 16-kbp het DNA fragment. We identified antigenic polypeptides produced in COS-1 cells after gene transfer of various cloned het DNA fragments. The 20-kbp fragment encoded a cytoplasmic antigen of about 95 kilodaltons (kDa). The 16-kbp fragment encoded antigens located in the nucleus, nuclear membrane, and cytoplasm. These were represented by several polypeptides, the most prominent of which were about 55, 52, and 36 kDa. The 36-kDa polypeptide was localized to a 2.7-kbp BamHI fragment which had homology to standard BamHI-W and BamHI-Z. Another polypeptide of 50 kDa found in the nucleus was mapped to the 7.1-kbp BamHI het DNA fragment which spans the EcoRI site linking the 20- and 16-kbp fragments of het DNA. Thus, HR-1 het DNA encodes several discrete polypeptide products, one or more of which could be responsible for the unusual biologic properties of the virus. The composition, regulation, and ultimately the expression of some of these products relative to standard EBV is probably altered by the genomic rearrangements of het DNA.
Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen
Authors: Lucher L A , Brackmann K H , Symington J S , Green M
Published: January 2020

The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the DNA sequence, whereas no methionine was released from the 39K T antigen during the first six cycles of Edman degradation. We propose that the short-lived 43K T antigen is the primary product of the 13S mRNA, the 266R T antigen; the somewhat more stable 42K T antigen is the primary product of the 12S mRNA, the 235R T antigen.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of two influenza A viruses from a pilot whale
Authors: Hinshaw V S , Bean W J , Geraci J , Fiorelli P , Early G , Webster R G
Published: January 2020

Influenza A viruses of the H13N2 and H13N9 subtypes were isolated from the lung and hilar node of a pilot whale. Serological, molecular, and biological analyses indicate that the whale isolates are closely related to the H13 influenza viruses from gulls.
Immune responses to isolated human cytomegalovirus envelope proteins
Authors: Gönczöl E , Hudecz F , Ianacone J , Dietzschold B , Starr S , Plotkin S A
Published: January 2020

A group of envelope proteins of human cytomegalovirus, gA protein (L. Pereira, M. Hoffman, M. Tatsuno, and D. Dondero, Virology 139:73-86, 1984; L. Pereira, p. 383-404, in B. Roizman, ed., The herpesviruses, vol. 3, 1985), and two protein mixtures (58,000-molecular-weight [58K]-66K and 130K-66K), separated by serial columns prepared with anti-gA immunoglobulin G from sera of immunized guinea pigs, induced neutralizing antibodies and a cellular immune response in the animals. The gA is a disulfide-linked protein complex consisting of high-molecular-weight (greater than 200K), 130K-150K, and 55K-58K proteins.
Epstein-Barr virus-specific DNA polymerase in virus-nonproducer Raji cells
Authors: Ooka T , Lenoir G M , Decaussin G , Bornkamm G W , Daillie J
Published: January 2020

Virus-nonproducer Raji cells, when induced to early antigen synthesis by 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate, showed an increase in DNA polymerase activity. This enzyme has the characteristics of a typical Epstein-Barr virus DNA polymerase with regard to chromatographical pattern and biological properties: it is eluted from DEAE-cellulose at 0.08 M NaCl, has a high salt resistance, is sensitive to phosphonoacetic acid and phosphonoformate, and shows a substrate preference for poly(dC)-oligo(dG12-18). The resistance of Epstein-Barr virus polymerase activity to aphidicolin is a property distinct from that of HSV DNA polymerase. Viral DNA polymerase activity increases in the absence of Epstein-Barr virus DNA replication, indicating that this enzyme is an early viral protein.
Purification of the Epstein-Barr virus/C3d complement receptor of human B lymphocytes: antigenic and functional properties of the purified protein
Authors: Nemerow G R , Siaw M F , Cooper N R
Published: January 2020

The Epstein-Barr virus/C3d receptor (CR2) of human B lymphocytes was purified to homogeneity from Raji cells by immunoaffinity chromatography. The average yield of the 145-kilodalton receptor was 400 pmol (50 micrograms) per 10(10) cells, representing an approximate 75% recovery. The isolated 145-kilodalton protein was antigenically and functionally intact as it reacted with several anti-CR2 monoclonal antibodies and bound purified Epstein-Barr virus and C3d,g. These findings with the purified molecule provide an unequivocal demonstration of the dual receptor functions of this protein.
Allosteric control of simian virus 40 T-antigen binding to viral origin DNA
Authors: Vogt B , Vakalopoulou E , Fanning E
Published: January 2020

Simian virus 40 (SV40) large tumor antigen (T antigen) possesses several biochemical activities localized in different domains of the protein. These activities include sequence-specific binding to two major sites, I and II, in the SV40 control region, ATPase, and nucleotide-binding activity. In the present communication, we present evidence that specific binding of immunopurified T antigen to SV40 DNA is markedly inhibited by low concentrations of ATP, dATP, GTP, and dGTP. The inhibition is reversible after removal of the nucleotide, suggesting that simple nucleotide binding rather than a covalent modification of T antigen in the presence of ATP is responsible for the inhibition. The results suggest that T antigen may assume two conformations, one active and one inactive in binding to the SV40 origin of replication. In the presence of purine nucleoside triphosphates, the inactive conformation is favored.
Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope
Authors: Bonino F , Heermann K H , Rizzetto M , Gerlich W H
Published: January 2020

Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.
Nuclease mechanism of the avian retrovirus pp32 endonuclease
Authors: Grandgenett D P , Vora A C , Swanstrom R , Olsen J C
Published: January 2020

In vivo, the inferred circular retrovirus DNA precursor to the provirus contains two long terminal repeats (LTRs) in tandem. We studied the site-specific nicking of supercoiled DNA that contains tandem copies of avian retrovirus LTR DNA in vitro by using purified avian myeloblastosis virus pp32 endonuclease, Mg2+, and viral DNA substrates containing different LTR circle junction sequences. The results confirmed our previous observation that the pp32 protein generates two nicks, one in either viral DNA strand, each 2 nucleotides from the circle junction site. The specificity of nicking by pp32 was unchanged over an eight-fold range of protein concentration and with different avian retrovirus LTR circle junction substrates. These data are consistent with models which propose a role for the endonuclease in removal of two nucleotides from the LTR termini on integration of viral DNA in vivo.
Localization of the feline sarcoma virus fgr gene product (P70gag-actin-fgr): association with the plasma membrane and detergent-insoluble matrix
Authors: Manger R , Rasheed S , Rohrschneider L
Published: January 2020

The v-fgr oncogene codes for a unique transforming protein (P70gag-actin-fgr) that contains virus-specific determinants and cell-derived sequences for both a tyrosine-specific kinase domain and an actin domain. We examined the subcellular distribution of the v-fgr protein by immunofluorescence microscopy and various cell fractionation techniques. By immunofluorescence, the v-fgr protein was localized in a diffuse cytoplasmic pattern within transformed cells. The v-fgr protein was not detectable at substratum adhesion sites. Crude membrane preparations (P100) obtained from fgr-transformed cells contained elevated levels of P70gag-actin-fgr. Further analysis of membranes on discontinous sucrose gradients revealed that P70gag-actin-fgr cofractionated with plasma membranes. Using an alternate method of fractionation, we found that the majority of the v-fgr protein remained with the insoluble matrix obtained by treating cells with a buffer containing Triton X-100. When membranes were similarly treated with detergent, nearly all of v-fgr protein remained with the residual insoluble matrix. These results suggest that the transforming activity of P70gag-actin-fgr may be directed to subcellular cytoskeletal targets at or near the cytoplasmic face of the plasma membrane.
In vitro mutagenesis of the putative membrane-binding domain of polyomavirus middle-T antigen
Authors: Markland W , Cheng S H , Oostra B A , Smith A E
Published: January 2020

Polyomavirus middle-T antigen contains a contiguous sequence of 22 hydrophobic amino acids near the carboxyl terminus, which is the putative membrane-binding domain of the protein. The DNA encoding this region was mutated to form a series of deletions, insertions, and substitutions called RX mutants. The phenotypes of these mutants fall into three groups based on the transforming and biochemical properties of their encoded proteins. The first group, with deletions outside but proximal to the hydrophobic domain, displayed an essentially wild-type phenotype. A second group, with extensive deletions within the region encoding the hydrophobic domain, expressed middle-T species which did not fractionate with cellular membranes or associate with pp60c-src and which were defective in their ability to transform. A third group of mutants with more subtle predicted alterations in the hydrophobic domain were wild type for the biochemical parameters investigated but were unable to transform cultured rodent cells. These observations are consistent with previous findings that membrane association plays an important role in transformation by middle-T and that, whereas association between middle-T and pp60c-src is a necessary correlate of transformation, it is not sufficient. A comparison of murine polyomavirus middle-T and a newly described hamster papovavirus putative middle-T revealed a strong homology between their respective hydrophobic-domain amino acid sequences. This homology is not observed in the anchorage domains of other model proteins, and this may imply that the middle-T hydrophobic domain is important in transformation for reasons other than simple membrane association.
A novel retroviruslike family in mouse DNA
Authors: Itin A , Keshet E
Published: January 2020

In the course of structural analysis of VL30 DNA elements, a recombinant retroviruslike element was encountered that contained non-VL30 long terminal repeats (LTRs) flanking internal VL30 sequences. With the aid of this novel LTR sequence probe, we cloned several DNA elements that were apparently members of a new retroviruslike family. A particular DNA element representative of this family (designated GLN) was characterized. It was approximately 8 kilobase pairs long and contained LTRs that are 430 base pairs long. It possessed an unusual primer-binding site sequence that corresponds to tRNAGln and a polypurine tract primer that is adjacent to the 3' LTR. The nucleotide sequences of the LTRs and their adjacent regions (which together housed all cis-acting retroviral functions) were different from those of known retroviruses and retroviruslike families. The comparison of three different GLN LTR sequences revealed a marked heterogeneity of U3 sequences relative to the homogeneity of R and U5 sequences. We estimate that approximately 20 to 50 copies of GLN elements are dispersed in all species of mice. GLN-related LTRs, however, are present in a much higher copy number (1,000 to 1,500 per genome). Nucleotide sequences that are more distantly related to GLN DNA are present in multiple copies in DNAs of other rodents but not in nonrodent genomes.
Structure and transforming function of transduced mutant alleles of the chicken c-myc gene
Authors: Patschinsky T , Jansen H W , Blöcker H , Frank R , Bister K
Published: January 2020

A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v-myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles.
Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence
Authors: Dalziel R G , Lampert P W , Talbot P J , Buchmeier M J
Published: January 2020

Strains of the murine coronavirus mouse hepatitis virus type 4 (MHV-4) which contained a mutation in the E2 peplomer glycoprotein were obtained by selection for resistance to neutralization by monoclonal antibodies. Characterization of six variants representing two independent epitopes on E2, E2B and E2C, by in vitro neutralization and antibody-binding assays demonstrated that selection for an alteration in epitope E2B also resulted in changes in epitope E2C and vice versa. We observed a mutation frequency of approximately 10(-4.3) to 10(-4.6), which is consistent with the expected occurrence of single point mutations. The variant virus strains were attenuated with respect to neurovirulence when compared with wild-type MHV-4. Mice normally develop encephalomyelitis and die after wild-type MHV-4 infection. Mice receiving 2- to 3-log-higher doses of the variant strains survived and developed demyelinating disease. As the disease progressed, evidence of remyelination and ongoing demyelination was observed up to 65 days after infection. Virus reisolated 15 days after infection retained the variant phenotype. The data indicate that the E2 glycoprotein plays a central role in determining the cellular tropism and virulence of MHV-4 in the mouse.
Mutation in the VP-1 gene is responsible for the extended host range of a monkey B-lymphotropic papovavirus mutant capable of growing in T-lymphoblastoid cells
Authors: Kanda T , Furuno A , Yoshiike K
Published: January 2020

Monkey B-lymphotropic papovavirus (LPV) replicates only in B-lymphoblastoid cells, whereas the LPV mutant 76 (LPV-76) can grow in either B- or T-lymphoblastoid cells. The nucleotide sequence of the wild-type LPV PstI B segment was compared with that of LPV-76 PstI-B, within which the mutation responsible for the extended host range had been located. The VP-1 coding region of LPV-76 was found to have mutations, single-base substitutions causing three amino acid substitutions.
Amplification and chromosomal dispersion of human endogenous retroviral sequences
Authors: Steele P E , Martin M A , Rabson A B , Bryan T , O'Brien S J
Published: January 2020

Endogenous retroviral sequences in humans have undergone amplification events involving both viral and flanking cellular sequences. We cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked.
Terminal structure and heterogeneity in human cytomegalovirus strain AD169
Authors: Tamashiro J C , Spector D H
Published: January 2020

We have characterized the heterogeneity occurring at the junction of the long (L) and short (S) segments and at the termini of the strain AD169 human cytomegalovirus (HCMV) genome by restriction endonuclease mapping and nucleotide sequence analyses. The HCMV a sequence was identified by its position at both termini and inverted orientation at the L-S junction. Heterogeneity at both termini and the L-S junction was generated by the presence of fused and tandem a sequences. Some S termini lacked an a sequence. In addition, near the L terminus and at the L-S junction there were a variable number of 217-base-pair (bp) XhoI fragments arranged in tandem. The 217-bp fragments consisted of a portion of the a and adjacent b sequences (in the L-segment repeat) bounded by the same direct repeats (DR1) found at the boundaries of the a sequence. A model for the generation of these heterogeneous fragments is presented. We also determined the sequence of seven cloned terminal fragments, five from the L terminus and two from the S terminus. All L termini contained identical terminal sequences ending with base 32 of a 33-bp DR1. The S termini differed from each other and from the L-segment termini. One S terminus lacked an a sequence and terminated within S-segment repeat (c) sequences. The second S terminus contained an a sequence and terminated with bases 20 to 33 of a 33-bp DR1. A comparison of the cloned L and S terminal sequences with cloned L-S junction sequences suggested that the termini contained 3' single base extensions which were removed during the cloning. We also show that the herpesvirus conserved sequence is in a similar position relative to the termini of HCMV and several other herpesviruses, thus adding further support for the role of the sequence in the maturation of viral DNA.
Expression of simian virus 40 early and late genes in mouse oocytes and embryos
Authors: Chalifour L E , Wirak D O , Wassarman P M , DePamphilis M L
Published: January 2020

Simian virus 40 (SV40) large- and small-tumor antigens (T-Ag, t-Ag) are normally synthesized early after infection of either permissive (monkey) or nonpermissive (mouse) fibroblasts, whereas an equivalent amount of viral coat protein (V-Ag) is observed late after infection of permissive cells and only after viral DNA replication has occurred. To determine whether or not expression of these genes is regulated in the same manner during early mammalian development, SV40 DNA was injected into the nuclei of mouse oocytes and one- and two-cell embryos. In oocytes, about three times more V-Ag was produced than T-Ag, and both were synthesized concomitantly in the same cells. Viral mRNA and proteins synthesized in oocytes comigrated during gel electrophoresis with the same products synthesized in SV40-infected monkey cells. Viral gene expression required circular DNA molecules injected into the nuclei of transcriptionally and translationally active cells. Injected DNA was stable and underwent conformational changes consistent with chromatin assembly. Oocytes did not replicate either polyomavirus or SV40 DNA. Thus, the temporal order of viral gene expression is circumvented in mouse germ cells, allowing these proteins to be expressed concurrently and in equivalent amounts with no requirement for DNA replication. However, in preimplantation embryos, neither T-Ag nor V-Ag was detected by immunoprecipitation although T-Ag synthesis was demonstrated as a specific requirement for SV40 DNA replication. Thus, viral gene expression in mouse embryos as early as the one-cell stage was reduced at least 500-fold relative to that in oocytes. Similarities between SV40 gene expression in mouse oocytes and that in Xenopus oocytes suggest that germ cells in higher animals share common regulatory mechanisms.