bluuebunny/crossref_metadata_2025 huggingface dataset:
Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation
Authors: Garcea R L , Ballmer-Hofer K , Benjamin T L
Published: January 2020

The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine.
Purification and properties of African swine fever virus
Authors: Carrascosa A L , del Val M , Santarén J F , Viñuela E
Published: January 2020

We describe a method for African swine fever (ASF) virus purification based on equilibrium centrifugation in Percoll density gradients of extracellular virions produced in infected VERO cells that yielded about 15 +/- 9% recovery of the starting infectious virus particles. The purified virus preparations were essentially free of a host membrane fraction (vesicles) that could not be separated from the virus by previously described purification methods. The purified virus sedimented as a single component in sucrose velocity gradients with a sedimentation coefficient of 3,500 +/- 300S, showed a DNA-protein ratio of 0.18 +/- 0.02 and a specific infectivity of 2.7 X 10(7) PFU/micrograms of protein, and remained fully infectious after storage at -70 degrees C for at least 7 months. The relative molecular weights of the 34 polypeptides detected in purified virus particles ranged from 10,000 to 150,000. Some of these proteins were probably cellular components that might account for the reactivity of purified virus with antiserum against VERO cells.
BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus
Authors: Adams R H , Brown D T
Published: January 2020

The process by which Sindbis virus excludes superinfecting homologous virus was investigated with the use of temperature-sensitive mutants. Mutants in two RNA-negative complementation groups were found to be defective in their ability to establish interference at the nonpermissive temperature. These mutants were unable to establish interference in a mixed infection (complementation), suggesting that both were defective in a common gene product. Homologous interference was found to block the replication of superinfecting virus after attachment, penetration, and translation of the nonstructural genes encoded in the virus RNA. The production of nonstructural gene products of superinfecting wild-type virus was found to enhance the replication of certain RNA- temperature-sensitive interfering viruses at the permissive and the nonpermissive temperature. The ability of certain RNA- mutants to establish homologous interference and to demonstrate enhanced growth after superinfection with wild-type virus was interpreted to produce a model implicating both virus and host components in the establishment of homologous interference and in the replication of Sindbis virus RNA.
Inhibition of binding to initiation complexes of nascent reovirus mRNA by double-stranded RNA-dependent protein kinase
Authors: De Benedetti A , Williams G J , Baglioni C
Published: January 2020

A coupled, cell-free system for the transcription and translation of reovirus mRNA was developed. Activated reovirions were incubated with reticulocyte lysate and an appropriate energy mix. Active transcription was obtained, but protein synthesis was inhibited after a short lag even by low concentrations of reovirions. This inhibition was abolished by the addition of the kinase inhibitor 2-aminopurine. With this addition, the synthesis of viral proteins could be detected in reaction mixtures containing nuclease-treated reticulocyte lysate. The binding of nascent reovirus mRNA to 80S initiation complexes measured after 2 min of incubation was greatly inhibited, whereas the binding of cellular mRNA added to the same reaction mixtures for the next 2 min was not inhibited. The inhibition of reovirus mRNA binding could not be explained by the synthesis of defective templates, since most of the mRNA could be bound to 80S complexes after the addition of 2-aminopurine. These results indicate that the binding of nascent reovirus mRNA was preferentially inhibited by a protein kinase. Reovirions preincubated with reticulocyte lysate could phosphorylate initiation factor eIF-2. This phosphorylation was inhibited by the addition of high concentrations of double-stranded RNA, which are inhibitory for the eIF-2 kinase present in elevated levels in reticulocyte lysate and in interferon-treated cells. These results indicate that the translation of viral mRNA may be preferentially inhibited in interferon-treated cells by the eIF-2 kinase activated by viral transcriptional complexes containing double-stranded RNA.
Retrovirus D/New England and its relation to Mason-Pfizer monkey virus
Authors: Desrosiers R C , Daniel M D , Butler C V , Schmidt D K , Letvin N L , Hunt R D , King N W , Barker C S , Hunter E
Published: January 2020

Seventeen isolates of retrovirus D/New England have been obtained from three species of macaques at the New England Regional Primate Research Center. Seven of the isolates were obtained from macaques who subsequently died with the macaque immunodeficiency syndrome; other isolates were obtained from macaques with less severe or other forms of illness. Attempts to isolate type D retrovirus from peripheral lymphocytes of 97 apparently healthy macaques have not been successful. Cloned DNA was prepared from Hirt supernatants of cells infected with one of these isolates (D/New England 398). By restriction endonuclease analysis, cloned pD398 DNA represented full-length viral DNA with one long terminal repeat. A detailed restriction endonuclease map of pD398 was derived and compared with a map of the cloned Mason-Pfizer monkey virus genome. Forty-six percent (13 of 28) of restriction endonuclease sites were found to be conserved when these related viruses were compared. Five of the D/New England isolates, including those from three different macaque species, were examined for strain variability by restriction endonuclease typing. Comparison of over 30 restriction endonuclease sites has not distinguished any of these D/New England isolates. It thus appears that a single strain of type D retrovirus is infecting three different species of macaques in the New England colony. Markedly reduced cross-hybridization was observed between cloned pD398 and Mason-Pfizer monkey virus DNAs at high stringency; this reduced cross-hybridization was localized to the pol-env regions of the genome. Only very weak hybridization of D/New England DNA to cloned squirrel monkey type D retrovirus DNA could be detected even at low-stringency conditions. What role type D retrovirus plays in the immunodeficiency syndrome of macaques remains to be determined.
Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis
Authors: Thomas D , Newcomb W W , Brown J C , Wall J S , Hainfeld J F , Trus B L , Steven A C
Published: January 2020

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.
A candidate for a new serotype of human rotavirus
Authors: Matsuno S , Hasegawa A , Mukoyama A , Inouye S
Published: January 2020

We investigated genetic and serological characteristics of a human rotavirus isolate from Indonesia which had a "super short" RNA electrophoretic pattern (A. Hasegawa, S. Inouye, S. Matsuno, K. Yamaoka, R. Eko, and W. Suharyono, Microbiol. Immunol. 28:719-722, 1984). This virus, strain 69M, was found by RNA-RNA hybridization to have a low degree of homology with the representative strains of all four human serotypes. Furthermore, it could not be classified by neutralization analysis into any of these serotypes. Therefore, this virus might belong to a new serotype.
The pX region of the bovine leukemia virus is transcribed as a 2.1-kilobase mRNA
Authors: Mamoun R Z , Astier-Gin T , Kettmann R , Deschamps J , Rebeyrotte N , Guillemain B J
Published: January 2020

The bovine leukemia virus mRNAs expressed in cultured bovine cells of various origins are a 9.0-kilobase genomic RNA, a 5.1-kilobase env RNA, and a newly detected 2.1-kilobase RNA corresponding to the transcription of pX sequences located in between the env gene and the 3' end of the provirus.
DNA sequence alterations responsible for the synthesis of thermosensitive VP1 in temperature-sensitive BC mutants of simian virus 40
Authors: Ng S C , Behm M , Bina M
Published: January 2020

The segment of simian virus 40 (SV40) genome which is recognized as the BC domain encodes for the COOH-terminal end of the SV40 major capsid protein VP1. Mutations in this domain lead to the synthesis of a thermosensitive VP1 which fails to assemble mature SV40 at the nonpermissive temperature. We determined the DNA sequences of eight BC mutants and compared them with the DNA sequences of wild-type SV40, polyomavirus, and BK virus. We found that BC11 and BC223 mutations result from changes in nucleotide residues 2367 (A to C) and 2084 (C to T), respectively. The others (i.e., BC208, BC214, BC216, BC217, BC248, and BC274) share the same point mutation at nucleotide 2354 (C to T). These mutations resulted in the following changes: Lys to Thr, His to Tyr, and Pro to Ser at VP1 amino acid residues 290, 196, and 286, respectively.
Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis
Authors: Gunn P R , Sato F , Powell K F , Bellamy A R , Napier J R , Harding D R , Hancock W S , Siegman L J , Both G W
Published: January 2020

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus.
Phytohemagglutinin activation of the transcription of the bovine leukemia virus genome requires de novo protein synthesis
Authors: Chatterjee R , Gupta P , Kashmiri S V , Ferrer J F
Published: January 2020

Addition of supramitogenic doses of phytohemagglutinin (PHA) to short-term cultures of neoplastic or nonneoplastic lymphocytes infected with bovine leukemia virus increased the synthesis of the major core virion antigen (p25) by 5- to 10-fold. Such stimulation was not due to the mitogenic effect of PHA or to a generalized increase in cellular RNA or protein synthesis but rather to enhanced transcription of the viral genome by a PHA-induced protein.
Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus
Authors: Oliff A , McKinney M D , Agranovsky O
Published: January 2020

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.
Insertional activation of c-myc by reticuloendotheliosis virus in chicken B lymphoma: nonrandom distribution and orientation of the proviruses
Authors: Swift R A , Shaller E , Witter R L , Kung H J
Published: January 2020

Chicken syncytial virus, a member of the reticuloendotheliosis virus family, can induce chicken B lymphomas indistinguishable from those caused by avian leukosis virus. Previously, we have demonstrated that the chicken syncytial virus proviruses in these tumors are linked to the proto-oncogene c-myc. We have now determined the arrangement of chicken syncytial virus proviruses in 22 tumors. The results indicate that these proviruses, without exception, are integrated upstream from the second c-myc exon. At least 70% of these insertion sites are clustered in a 0.5-kilobase region immediately preceding the exon. The proviruses are all arranged in the same transcriptional orientation as c-myc. This type of provirus organization bears strong resemblance to that of the avian leukosis virus proviruses involved in c-myc activation.
The immediate-early enhancer element of herpes simplex virus type 1 can replace a regulatory region of the c-Ha-ras1 oncogene required for transformation
Authors: Puga A , Gomez-Marquez J , Brayton P R , Cantin E M , Long L K , Barbacid M , Notkins A L
Published: January 2020

A 0.8-kilobase SacI DNA fragment in the distal 5'-noncoding region of the c-Ha-ras1 oncogene hybridized to high guanine X cytosine sites of herpes simplex virus type 1 (HSV-1) DNA restriction fragments. Nucleotide sequence comparisons localized one of these sites to the intergenic region of HSV between the immediate-early genes coding for IEmRNA-3 and IEmRNA-4/5 that has enhancer-type activity. We tested the possibility that the HSV-1 enhancer and the upstream c-Ha-ras1 SacI fragment were functionally related by assaying for the capacity of recombinant plasmids in which the HSV-1 enhancer replaced the oncogene 0.8-kilobase SacI fragment to transform NIH/3T3 cells. Deletion of the 0.8-kilobase SacI fragment abolished the biological activity of c-Ha-ras1, but its replacement by the HSV-1 enhancer fully restored it. These results confirm the enhancer properties of the HSV-1 immediate-early intergenic region and suggest that c-Ha-ras1 sequences contained within the 0.8-kilobase SacI fragment plays a role in the transcriptional activation of the oncogene.
Spontaneous temperature-sensitive mutations in bacteriophage T7
Authors: Stone J C , Miller R C
Published: January 2020

Attempts to recover temperature-sensitive mutations affecting genes 13 and 14 (virion proteins) in bacteriophage T7 by analysis of amber revertants were confounded by the frequent occurrence of spontaneous temperature-sensitive mutations in other genes. These incidental temperature-sensitive mutations are physically distinct from but may be functionally related to genes 13 and 14, as shown by complementation and recombination studies. The possibility that these incidental temperature-sensitive mutations represent secondary-site suppressors of the pseudonormal suppressed amber products is discussed.
Nucleotide sequence of a transduced myc gene from a defective feline leukemia provirus
Authors: Braun M J , Deininger P L , Casey J W
Published: January 2020

The nucleotide sequence of a feline v-myc gene and feline leukemia virus (FeLV) flanking regions was determined. Both the nucleotide and predicted amino acid sequences are very similar to the murine and human c-myc genes (ca. 90% identity). The entire c-myc coding sequence is represented in feline v-myc and replaces portions of the gag and env genes and the entire pol gene. The coding sequence is in phase with the gag gene reading frame; v-myc, therefore, appears to be expressed as a gag-myc fusion protein. Viral sequences at the 3' myc-FeLV junction begin with the hexanucleotide CTCCTC, which is also found at the 3' fes-FeLV junction of both Gardner-Arnstein and Snyder-Theilen feline sarcoma viruses. These similarities suggest that some sequence specificity may exist for the transduction of cellular genes by FeLV. Feline v-myc lacks a potential phosphorylation site at amino acid 343 in the putative DNA-binding domain, whereas both human and murine c-myc have such sites. Avian v-myc has lost a potential phosphorylation site which is present in avian c-myc five amino acids from the potential mammalian site. If these sites are actually phosphorylated in normal c-myc proteins, their loss may alter the DNA-binding affinity of v-myc proteins.
Transformation of Brown Leghorn chicken embryo fibroblasts by avian myeloblastosis virus proviral DNA
Authors: Soret J , Kryceve-Martinerie C , Crochet J , Perbal B
Published: January 2020

Brown Leghorn chicken embryo fibroblasts were transfected with a mixture of avian myeloblastosis virus (AMV) and myeloblastosis-associated virus type 1 (MAV1) proviral DNA purified from lambda-Charon 4A recombinant clones. A transformed cell line (T1AM) able to grow without anchorage in semisolid medium was obtained. The presence of both proviral AMV and MAV sequences was detected in T1AM DNA by hybridization with v-myb- and MAV1-specific probes. Altered AMV and MAV1 proviral genomes were found in T1AM genome. Characterization of the RNA species expressed in transformed cells showed that in addition to a 2.5-kilobase (kb) putative subgenomic v-myb-specific RNA, three other myb-containing RNAs (9.4, 8.4, and 7.0 kb) were present in T1AM cells. No AMV genomic RNA was detected. Also, a new 5.0-kb MAV1-specific RNA species was expressed in transformed cells in addition to MAV1 genomic RNA species (7.8 kb). No infectious AMV virions are released by T1AM cells. Chicken embryo fibroblasts infected by T1AM-released virions contained and expressed all MAV1 sequences detected in T1AM transformed cells but did not express any transformation parameter. These results indicated that the presence of AMV proviral sequences in T1AM cells is responsible for their transformed phenotype.
DNA-binding proteins present in varicella-zoster virus-infected cells
Authors: Roberts C R , Weir A C , Hay J , Straus S E , Ruyechan W T
Published: January 2020

DNA-binding proteins present in varicella-zoster virus-infected cells were identified by DNA-cellulose chromatography of radioactively labeled cell extracts. Seven virus-specific proteins, ranging in molecular weight from approximately 175,000 to 21,000, showed affinity for single- or double-stranded DNA or both. These proteins include the varicella-zoster virus major capsid protein, a phosphorylated tegument protein, and a 125,000-molecular-weight species which may be analogous to the major DNA-binding protein of herpes simplex virus. We also identified a number of DNA-binding phosphoproteins by these procedures. Finally, protein blot studies were carried out to determine whether these proteins bind preferentially to virus rather than to host cell DNA.
Evolutionary variants of Rous sarcoma virus: large deletion mutants do not result from homologous recombination
Authors: Voynow S L , Coffin J M
Published: January 2020

Large deletion (LD) mutants of Prague strain Rous sarcoma virus, subgroup B (PrB), derived by serial undiluted passage through chicken (C/E) cells, were isolated and characterized. Individual LD viruses were initially isolated by cloning in soft agar of infected, chemically transformed quail (QT6) cells. Two regions of the PrB genome were deleted in the formation of the LD virus. This resulted in the junction of gag sequences in p12 to env sequences in gp37, and in the loss of the src gene. DNA restriction analysis of biologically active lambda Charon 27-LD recombinant clones indicated that individual LD viruses contained similar but not identical deletion endpoints. Two LD isolates, LD25 and LD85, were further subcloned into pBR322, and the deletion junctions were examined by DNA sequencing. Although the gag-env deletion endpoints were identical in the two subclones, heterogeneity was observed across the src deletion in that both mutants analyzed had the same 5' endpoint but slightly different 3' endpoints. In all cases, only a single homologous base (always an A residue) was found at the deletion endpoint. S1 nuclease analysis of the RNA from a number of QT6-LD clones gave similar results, indicating that the LD population was composed of viruses with similar but not identical deletion endpoints. Such viruses may have been generated from errors during reverse transcription of the virion RNA with subsequent selection assuring their dominance in the population.
KBSH parvovirus: comparison with porcine parvovirus
Authors: Molitor T W , Joo H S , Collett M S
Published: January 2020

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.
Monoclonal antibodies to the M protein of vesicular stomatitis virus (Indiana serotype) and to a cDNA M gene expression product
Authors: Pal R , Grinnell B W , Snyder R M , Wiener J R , Volk W A , Wagner R R
Published: January 2020

Twenty-nine independent hybridomas producing monoclonal antibodies to the matrix (M) protein of vesicular stomatitis virus (Indiana serotype) were prepared by fusion of SP2/0 myeloma cells with spleen lymphocytes obtained from BALB/c mice which had been immunized with the purified M protein. The specific reactivity of each monoclonal antibody was determined by an enzyme-linked immunosorbent assay and a competitive binding assay. Most of the antibodies were of the immunoglobulin G2a and G2b isotypes, although some were immunoglobulin M. By measuring the competitive binding of 125I-antibody, we identified four antigenic determinants in the M protein of the virus; two of these determinants, however, exhibited a large degree of overlap. Western blot analysis revealed little or no cross-reactivity of the antibodies with other viral proteins or with the M protein of the New Jersey serotype. Prolonged trypsin proteolysis removed the first 43 amino acids from the amino-terminal region of the M protein, but it retained its reactivity with monoclonal antibodies to each epitope, except for diminished reactivity with one. To aid in future mapping of these epitopes, we inserted a cDNA clone of the mRNA encoding the M protein of vesicular stomatitis virus into an inducible lac expression vector; the M protein produced in the JM103 strain of Escherichia coli under induced conditions was found to be approximately the same size as native M protein and was recognized by the monoclonal antibodies. These monoclonal antibodies and the cDNA clone should be useful for studying the role of M protein in virus maturation and the regulation of viral transcription.
Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice
Authors: Sears A E , Halliburton I W , Meignier B , Silver S , Roizman B
Published: January 2020

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.
Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24
Authors: Casey J M , Kim Y , Andersen P R , Watson K F , Fox J L , Devare S G
Published: January 2020

The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.
Diagnosis of rotavirus infection with cloned cDNA copies of viral genome segments
Authors: Lin M , Imai M , Bellamy A R , Ikegami N , Furuichi Y , Summers D , Nuss D L , Deibel R
Published: January 2020

The diagnostic potential of cloned cDNA copies of human rotavirus (strain WA) genome segments for the detection of rotavirus in clinical specimens has been determined. A hybridization assay in which a mixture of 32P-labeled cDNAs representing the 11 rotavirus segments was used as a probe compared favorably with three frequently used diagnostic tests for rotavirus in terms of both specificity and sensitivity. Significantly, clinical isolates could be readily distinguished when cloned cDNA copies of individual genome segments were used independently as a probe. In assays in which genome RNA from rotaviruses of known subgroups and serotypes were tested, cloned probes that encode nonstructural viral proteins hybridized efficiently to genome RNAs of all strains, whereas cloned probes corresponding to genome segments 6 and 9 exhibited the potential for differentiating strains of different subgroups and serotypes. Cloned cDNA copies of rotavirus genome segments therefore offer considerable potential for improved general diagnosis of rotavirus in clinical specimens, as well as for epidemiological studies in which virus isolates can be distinguished on the basis of nucleotide sequence homology of individual genome segments.
Development of resistance of enterocytes to rotavirus in neonatal, agammaglobulinemic piglets
Authors: Kirstein C G , Clare D A , Lecce J G
Published: January 2020

The first part of this report describes the development of a technique for evaluating the growth of rotavirus under controlled conditions that approximate a natural infection. A standard dose of rotavirus (approximately 10(9) viral particles) was injected into ligated segments in the small intestine of newborn, agammaglobulinemic, colostrum-deprived piglets. After various periods postinoculation, the segments were retrieved and the enterocytes were evaluated for the presence of rotaviral antigens by immunofluorescence and rotaviral particles by transmission electron microscopy. Peak immunofluorescence in enterocytes was detected at 8 h postinoculation in the upper and middle jejunum and ileum. Transmission electron microscopy at this time revealed fully formed virions which were not seen in sections examined before this 8-h period. The second part of our study describes the use of ligated segments in determining the susceptibility to rotavirus of enterocytes in piglets ranging in age from newborn to 2 weeks. By the time piglets were 2 days old, enterocytes in the upper half of the small intestines appeared to be resistant to rotavirus, whereas those in the lower half seemed partially resistant. Between 4 and 8 days of age, enterocytes in the lower half also became resistant. Resistance paralleled the loss in capacity of piglets to transport macromolecules through enterocytes and was not correlated with the loss in capacity to internalize macromolecules.
Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase
Authors: Morrison D K , Carter J K , Moyer R W
Published: January 2020

A library of monoclonal antibodies directed against individual proteins of the rabbit poxvirus (RPV) virion within a complex immunogenic mixture has been generated through the use of in vivo and in vitro immunization regimens. The relative efficacies of the two procedures were compared. Based on immunoblot analysis, the in vitro immunization regimen led both to a wider variety of monoclonal antibodies to different proteins and to a larger number of antibodies directed against proteins of higher molecular weights. Each method, however, has advantages, and the two procedures appear to be complementary. A simple method to recognize antibodies directed against the virion DNA-directed RNA polymerase was developed. Monoclonal antibodies directed against two subunits (137 and 34 kilodaltons [kDa]) of the RNA polymerase were identified and used to study the biogenesis of the enzyme and to map the two corresponding genes within the viral genome by using an RPV DNA library cloned into the lambda gtll expression vector. Both proteins are synthesized late in the infectious cycle and are restricted totally to the cytoplasm. Preliminary mapping data place the genes encoding the 137-kDa protein within the HindIII H fragment, whereas the gene for the 34-kDa protein is located within the left most region of the HindIII A fragment.
Epstein-Barr virus-encoded protein found in plasma membranes of transformed cells
Authors: Mann K P , Staunton D , Thorley-Lawson D A
Published: January 2020

We have developed monoclonal antibodies to a 63,000-molecular-weight protein (p63) which is the product of the most abundant messenger RNA in Epstein-Barr virus-transformed cells and shown that the protein is associated specifically with plasma membranes. It was also found to be associated with the other membrane fractions and was found in all Epstein-Barr virus-transformed cells tested. In addition, p63 was present in virions, resulting in transient, early appearance in newly infected cells. Newly synthesized p63 was detected at the time cells underwent blast transformation (48 to 72 h postinfection). The possible role of this protein in transformation and as a target for cell-mediated cytotoxicity is discussed.
Correspondence between immunological and functional domains in the transforming protein of Fujinami sarcoma virus
Authors: Stone J C , Pawson T
Published: January 2020

Monoclonal antibodies reactive with either gag or fps portions of the wild-type Fujinami sarcoma virus transforming protein have been used to probe the structure of proteins encoded by mutant genomes constructed in vitro. The pattern of immunoreactivity suggests that the functional domain defined in genetic studies (Stone et al., Cell 37:549-558, 1984) corresponds to a discrete immunological domain in the native, wild-type Fujinami sarcoma virus protein. At least one mutation affecting both the structure and function of the proposed NH2-terminal fps-specific domain encodes a product with high specific activities in kinase assays. Furthermore, a cell line expressing high levels of this mutant protein is only moderately transformed. The striking correspondence between the immunological domain defined here and the functional domain inferred from the results of transfection experiments suggests that this non-kinase-specifying region constitutes a discrete structural as well as functional component of the viral protein.
ts1, a Paralytogenic mutant of Moloney murine leukemia virus TB, has an enhanced ability to replicate in the central nervous system and primary nerve cell culture
Authors: Wong P K , Knupp C , Yuen P H , Soong M M , Zachary J F , Tompkins W A
Published: January 2020

A temperature-sensitive mutant of Moloney murine leukemia virus TB (MoMuLV-TB), ts1, which is defective in intracellular processing of envelope precursor protein (Pr80env), also possesses the ability to induce hind-limb paralysis in infected mice. To investigate whether ts1 has acquired neurotropism and to determine to what extent it can replicate in the central nervous system, we compared viral titers in the spleen, plasma, spinal cord, and brain throughout the course of infection of mice infected with ts1 and parental wild-type (wt) MoMuLV-TB. In both the ts1- and wt-inoculated mice, the concentrations of infectious virus recovered from the plasma and spleen increased rapidly and reached a plateau by 10 days postinfection (p.i.). In contrast, virus concentrations in the spinal cord and brain of ts1-inoculated mice increased gradually and reached a titer comparable to that in the spleen and exceeding that in the plasma only at 25 to 30 days p.i. At this time, the virus titer was approximately 200X greater in ts1-infected spinal cord tissue and approximately 20X greater in ts1-infected brain tissue than in the same wt-infected tissues. Paralysis became evident at 25 to 30 days p.i. in ts1-inoculated mice, whereas the wt-inoculated mice were normal. In addition, a substantial amount of Pr80env was detected in the spinal cords of ts1-inoculated mice compared with that found in the spinal cords of wt-inoculated mice. The infectious virus isolated from ts1-infected nerve tissue was found to possess the characteristic phenotype of the ts1 virus. Microscopic lesions of ts1-inoculated mice at 30 days p.i. consisted of vacuolar degeneration of motor neurons and spongy change of white matter in the brain stem and spinal cord. Similar but less severe lesions were observed in wt-inoculated mice. With primary cultures of central nervous system tissue we showed that ts1 can infect and replicate in both neuron and glial cells. In contrast, although wt MoMuLV-TB replicated in glial cell-rich culture, viral replication was barely detectable in neuron-rich culture.
Unusual features of the leader sequence of Rous sarcoma virus packaging mutant TK15
Authors: Nishizawa M , Koyama T , Kawai S
Published: January 2020

TK15, a mutant derived from a temperature-sensitive mutant of Rous sarcoma virus (tsNY68), has extremely low infectivity although it has intact viral genes. Biological and biochemical analyses of the virus and virus-induced transformants showed that the mutant has a defect in the packaging of its own genomic RNA possibly owing to a deletion near the 5' end (S. Kawai and T. Koyama, J. Virol. 51:147-153, 1984; T. Koyama, F. Harada, and S. Kawai, J. Virol. 51:154-162, 1984). Nucleotide sequence analysis of the provirus DNA of the mutant revealed that the deletion extends from the 3' end of the primer binding site to 22 bases upstream of the gag initiation codon and also suggested that possible binding between an extra region of the primer molecule and the viral genome resulting from the deletion causes another defect in the replication of the TK15 genome. It was suggested that the deletion of 237 bases in TK15 was generated during reverse transcription of the genome by the skipping of a sequence between identical 13-base sequences present in the primer binding site and 35 to 22 bases upstream of the gag initiation codon of the parental virus.
trans-Activation of parvovirus P38 promoter by the 76K noncapsid protein
Authors: Rhode S L
Published: January 2020

The autonomously replicating parvoviruses contain a 5-kilobase linear single-stranded DNA genome that produces two noncapsid proteins, N1 and N2, and two overlapping capsid proteins, VP1 and VP2. To characterize the regulation of viral transcription, we began with a study of the promoter for the coat proteins (P38) at map unit 38. Various constructions containing the P38 promoter were fused to the bacterial gene for chloramphenicol acetyltransferase (cat), and the relative efficiency of expression was determined in the presence and absence of parvovirus gene products. Our results show that the P38 promoter is a weak promoter without a trans-activation mediated by the 76,000-molecular-weight (76K) N1 protein. The N1 protein, supplied either by superinfection with virus or cotransfection with the cloned N1 gene, increased greatly the expression of the P38 promoter. In addition, sequences 3' to the promoter, within the region + 127 to + 648 (assuming an mRNA start site at 2008), were required for optimal expression but not for trans-activation. These results suggest that the production of parvovirus capsid proteins is under the indirect control of the P4 promoter and one of its gene products.
Regulation of herpes simplex virus-specific lymphoproliferation by suppressor cells
Authors: Horohov D W , Moore R N , Rouse B T
Published: January 2020

We investigated the regulation of the herpes simplex virus (HSV)-specific lymphoproliferative response (LPR) by suppressor cells. The chief cell types in HSV-immune splenocytes proliferating in response to the antigen were Lyt 1+ and Lyt 2+ T cells, which accounted for approximately 60 and 40% of the response, respectively. Because the total responsiveness of splenocytes was enhanced after depletion of Lyt 2+ cells, the LPR was assumed to be subject to regulation by an Lyt 2+ suppressor cell. This was shown to be the case with an experimental design in which suppressor cell activity was induced in one culture, the cells were irradiated, and the effects on LPR were measured in a test antigen-stimulated culture. The cell responsible for suppression was shown to be Lyt 2+ IJ+, and the actual suppressor effect was not antigen specific. Cellular requirements for the generation of suppression were also investigated. The three distinct cell types that appeared to be required were Lyt 2+ and Lyt 1+ T cells and an IJ+ antigen-presenting cell. Of the three cell types, only the Lyt 2+ cell needed to be from HSV-immune animals. The implications of our model system for the better understanding of the role of immunity in herpesvirus pathogenesis are discussed.
Origin binding by a 100,000-dalton super-T antigen from SVT2 cells
Authors: Wilson V G , Williams R L
Published: January 2020

The SVT2 line of simian virus 40-transformed mouse cells expresses little or no wild-type-size A protein (T antigen). Instead, a variant form is produced in these cells that is larger than normal-size A protein. This variant form has an Mr of 100,000 (100K super-T antigen) and is found primarily in complexes with the host-cell-coded p53 protein. Binding of the 100K super-T antigen to simian virus 40 origin region DNA was assayed by immunoprecipitation of super-T antigen-DNA complexes and then digestion with DNase I. DNA sequences associated with super-T antigen were protected from digestion and retained in the immune complex, while unprotected sequences were digested and released. The 100K super-T antigen efficiently protects DNA sequences in the previously defined regions I and II (P. Tegtmeyer, B. A. Lewton, A. L. DeLucia, V. G. Wilson, and K. Ryder, J. Virol. 46:151-161, 1983). Within region II (the origin of replication), the pattern and size of protected fragments are identical for super-T antigen and purified wild-type A protein. Thus, even though super-T antigen is larger than wild-type A protein, both must bind with the same alignment on origin DNA. Furthermore, complexes between the host-cell-coded p53 protein and the 100K super-T antigen also retain the ability to bind in regions I and II.