bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombination between two integrated proviruses, one of which was inserted near c-myc in a retrovirus-induced rat thymoma: implications for tumor progression
Authors: Lazo P A , Tsichlis P N
Published: January 2020

Of 17 Moloney murine leukemia virus (MoMuLV)-induced rat thymomas, 2 contained rearrangements in c-myc. In one of these tumors the observed rearrangement was not due to the insertion of an intact MoMuLV provirus. The rearranged c-myc DNA fragment from this thymoma was cloned and examined by restriction endonuclease mapping, hybridization to MoMuLV proviral DNA probes, and DNA sequence analysis. These analyses revealed that the c-myc rearrangement in this tumor was due to the presence of a partially duplicated MoMuLV long terminal repeat (LTR) 5' to c-myc exon 1. The orientation of this LTR structure was opposite to the transcriptional orientation of c-myc. The sequences at the 3' flanking side of the LTR structure were derived from a cellular DNA region which maps to the same chromosome as c-myc (chromosome 7), although to a site distant from this proto-oncogene. These findings present evidence for a homologous recombination event occurring between sequences of two proviruses integrated on the same chromosome, one of which was inserted near the c-myc proto-oncogene. The recombination product contains three copies of the MoMuLV LTR 72-base-pair direct repeat and is associated with a high level of c-myc expression. The reciprocal product of this recombination was not detected. We propose that recombination between homologous sequences may play a significant role in the generation of chromosomal rearrangements and therefore in tumor induction and progression.
Comparative molecular genetic analysis of lymphomas from six inbred mouse strains
Authors: Mucenski M L , Bedigian H G , Shull M M , Copeland N G , Jenkins N A
Published: January 2020

Previous studies of 21 highly lymphomatous AKXD recombinant inbred mouse strains demonstrated correlations between lymphoma type, the somatic proviral DNA content of the lymphoma, and the frequency of virally induced rearrangements in eight common sites of viral integration (Myc, Pim-i, Pvt-1, Mlvi-1, Mlvi-2, Fis-1, Myb, and Evi-1). In this study we analyzed lymphomas from six inbred mouse strains, AKR/J, C58/J, HRS/J (hr/hr and hr/+), SJL/J, SEA/GnJ, and CWD/LeAgl, to determine whether these correlations are also evident in these strains. Mice of the AKR/J, C58/J, and HRS/J strains died exclusively of T-cell lymphomas. In contrast to earlier studies which showed a great disparity in the rate and incidence of lymphomas in HRS/J hr/hr and HRS/J hr/+ mice, we found a high incidence of T-cell lymphomas and the same mean age of onset of disease in both strains. SJL/J mice died primarily of pre-B-cell lymphomas, whereas CWD/LeAgl and SEA/GnJ mice died primarily of B-cell lymphomas. Somatically acquired mink cell focus-forming proviruses were detected only in T-cell lymphomas, whereas ecotropic proviruses were found in lymphomas from all hematopoietic cell lineages. No rearrangements were detected in the Fis-1, Mlvi-2, and Myb loci, whereas rearrangements were detected in the Mlvi-1, Myc, Pim-1, Pvt-1, and Evi-1 loci. Most rearrangements were found in T-cell lymphomas, and many were virally induced. These results are similar to those we obtained previously for lymphomas of 21 highly lymphomatous AKXD recombinant inbred mouse strains.
Different patterns of Epstein-Barr virus gene expression and of cytotoxic T-cell recognition in B-cell lines infected with transforming (B95.8) or nontransforming (P3HR1) virus strains
Authors: Murray R J , Young L S , Calender A , Gregory C D , Rowe M , Lenoir G M , Rickinson A B
Published: January 2020

Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines have been converted to EBV genome positivity by in vitro infection with the transforming EBV strain B95.8 and with the nontransforming mutant strain P3HR1, which has a deletion in the gene encoding the nuclear antigen EBNA2. These B95.8- and P3HR1-converted lines have been compared for their patterns of expression of EBV latent genes (i.e., those viral genes constitutively expressed in all EBV-transformed lines of normal B-cell origin) and for their recognition by EBV-specific cytotoxic T lymphocytes (CTLs), in an effort to identify which latent gene products provide target antigens for the T-cell response. B95.8-converted lines on several different EBV-negative BL-cell backgrounds all showed detectable expression of the nuclear antigens EBNA1, EBNA2, and EBNA3 and of the latent membrane protein (LMP); such converts were also clearly recognized by EBV-specific CTL preparations with restriction through selected human leukocyte antigen (HLA) class I antigens on the target cell surface. The corresponding P3HR1-converted lines (lacking an EBNA2 gene) expressed EBNA1 and EBNA3 but, surprisingly, showed no detectable LMP; furthermore, these converts were not recognized by EBV-specific CTLs. Such differences in T-cell recognition were not due to any differences in expression of the relevant HLA-restricting determinants between the two types of convert, as shown by binding of specific monoclonal antibodies and by the susceptibility of both B95.8 and P3HR1 converts to allospecific CTLs directed against these same HLA molecules. The results suggest that in the normal infectious cycle, EBNA2 may be required for subsequent expression of LMP and that both EBNA2 and LMP (but not EBNA1 or EBNA3) may provide target antigens for the EBV-specific T-cell response.
Herpes simplex virus virion host shutoff function
Authors: Kwong A D , Kruper J A , Frenkel N
Published: January 2020

Herpes simplex virus (HSV) virions contain one or more functions which mediate the shutoff of host protein synthesis and the degradation of host mRNA. HSV type 1 (HSV-1) mutants deficient in the virion shutoff of host protein synthesis (vhs mutants) were isolated and were found to be defective in their ability to degrade host mRNA. Furthermore, it was found that viral mRNAs in cells infected with the vhs 1 mutant have a significantly longer functional half-life than viral mRNAs in wild-type virus-infected cells. In the present study we have mapped the vhs1 mutation affecting the virion shutoff of host protein synthesis to a 265-base-pair NruI-XmaIII fragment spanning map coordinates 0.604 to 0.606 of the HSV-1 genome. The mutation(s) affecting the functional half-lives of host mRNA as well as the alpha (immediate-early), beta (early), and gamma (late) viral mRNAs were also mapped within this 265-base-pair fragment. Thus, the shutoff of host protein synthesis is most likely mediated by the same function which decreases the half-life of viral mRNA. The shorter half-life of infected-cell mRNAs may allow a more rapid modulation of viral gene expression in response to changes in the transcription of viral genes. Interestingly, the vhs1 mutation of HSV-1 maps within a region which overlaps the Bg/II-N sequences of HSV-2 DNA shown previously to transform cells in culture. The possible relationship between the transformation and host shutoff functions are discussed.
trans-acting regulation of bovine leukemia virus mRNA processing
Authors: Derse D
Published: January 2020

Bovine leukemia virus (BLV) and the human T-cell leukemia virus types I and II comprise a unique retrovirus subfamily which has evolved complex strategies for the regulation of gene expression. A transcriptional control circuit has been characterized in both human and bovine systems in which cis-acting promoter control elements are responsive to trans-acting factors encoded in the pX region of the virus. The BLV pX mRNA encoding the transcriptional trans-acting factor is translated in an alternate reading frame to produce an 18-kilodalton nuclear phosphoprotein, p18. A function for this protein was revealed in cotransfection experiments using mutated BLV proviruses in combination with pX expression plasmids. These experiments indicated that p18 was required for the accumulation of viral mRNAs representing full-length (genomic) and single-spliced (env) transcripts. In contrast, synthesis of the double-spliced pX mRNA was not influenced by p18 expression. Large regional deletions and substitutions of provirus sequences localized elements essential for p18 regulation to the 3' long terminal repeat. Furthermore, sequences within a 250-nucleotide region between the AATAAA signal and poly(A) site were found to be essential for efficient virus mRNA 3'-end processing and response to p18 regulation.
A cowpox virus gene required for multiplication in Chinese hamster ovary cells
Authors: Spehner D , Gillard S , Drillien R , Kirn A
Published: January 2020

Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus.
A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells
Authors: Ligas M W , Johnson D C
Published: January 2020

Herpes simplex virus (HSV) glycoprotein gD is a major component of the virion envelope and is thought to play an important role in the initial stages of viral infection and stimulates the production of high titers of neutralizing antibodies. We assumed that gD plays an essential role in virus replication, and so to complement viruses with mutations in the gD gene we constructed a cell line, denoted VD60, which is capable of expressing high levels of gD after infection with HSV. A recombinant virus, designated F-gD beta, in which sequences encoding gD and a nonessential glycoprotein, gI, were replaced by Escherichia coli beta-galactosidase sequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers under agarose overlays containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). F-gD beta was able to replicate normally on complementing VD60 cells. However, F-gD beta was unable to form plaques on noncomplementing Vero cells. Virions lacking gD were produced in normal amounts by Vero cells infected with F-gD beta, and the virus particles were distributed throughout the cytoplasm and on the cell surface, suggesting that gD is not essential for HSV envelopment and egress. Virions lacking gD were able to bind to cells, but were unable to initiate synthesis of viral early polypeptides. Plaque production of F-gD beta particles lacking gD was enhanced by polyethylene glycol treatment, suggesting that gD is essential for penetration of HSV into cells. Other HSV glycoproteins have been implicated in the entry of virus into cells, and thus this process appears to involve multiple interactions at the cell surface.
Evidence of restricted viral replication in adult mink infected with Aleutian disease of mink parvovirus
Authors: Alexandersen S , Bloom M E , Wolfinbarger J
Published: January 2020

Strand-specific hybridization probes were used in in situ molecular hybridization specifically to localize cells containing replicative intermediates of Aleutian disease of mink parvovirus (ADV). When adult mink of Aleutian genotype were infected with ADV Utah I, the largest number of cells positive for viral replication (i.e., containing replicative-form DNA and RNA) were found in the mesenteric lymph nodes and spleens at 10 days after infection. The localization of positive cells in the middle of germinal centers suggested that they were B lymphoblasts. Circulating leukocytes and bone marrow cells also contained viral RNA, but the levels of replicative-form DNA were below detectability. The levels of viral DNA and RNA in adult mink cells replicating ADV were decreased compared with those in permissively infected cell cultures or neonatal mink, suggesting that the replication of ADV in adult mink might be semipermissive or restricted at some early stage of viral gene expression. The low level of viral replication and transcription in lymphoid cells might provide a mechanism for the development of immune disorders and for the maintenance of persistent infection. Single-stranded virion DNA was found in other organs, but the strand-specific probes made it possible to show that this DNA represented virus sequestration. In addition, glomerular immune complexes containing virion DNA were detected, suggesting that ADV virions, or perhaps free DNA, may have a role in the development of ADV-induced glomerulonephritis.
Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection
Authors: Belshe R B , Smith M H , Hall C B , Betts R , Hay A J
Published: January 2020

The emergence of influenza A viruses which had acquired resistance to rimantadine during a clinical trial (C. B. Hall, R. Dolin, C. L. Gala, D. M. Markovitz, Y. Q. Zhang, P. H. Madore, F. A. Disney, W. B. Talpey, J. L. Green, A. B. Francis, and M. E. Pichichero, Pediatrics 80:275-282, 1987) provided the opportunity to determine the genetic basis of this phenomenon. Analysis of reassortant viruses generated with a resistant clinical isolate (H3N2) and the susceptible influenza A/Singapore/57 (H2N2) virus indicated that RNA segment 7 coding for matrix and M2 proteins conferred the resistant phenotype. Resistant viruses isolated from seven patients each contained a single change in the nucleotide sequence coding for the M2 protein which resulted in substitutions in amino acid 30 (two viruses) or 31 (five viruses) in the transmembrane domain of the molecule. These changes occurred in locations identified in influenza viruses selected for resistance to amantadine in tissue culture and indicate a common mechanism of action of the two compounds in cell culture and during chemotherapeutic use.
Transformation of human fibroblasts by ionizing radiation, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H-1 virus and minute virus of mice
Authors: Cornelis J J , Becquart P , Duponchel N , Salomé N , Avalosse B L , Namba M , Rommelaere J
Published: January 2020

Morphologically altered and established human fibroblasts, obtained either by 60Co gamma irradiation, treatment with the carcinogen 4-nitroquinoline 1-oxide, or simian virus 40 (SV40) infection, were compared with their normal finite-life parental strains for susceptibility to the autonomous parvoviruses H-1 virus and the prototype strain of minute virus of mice (MVMp). All transformed cells suffered greater virus-induced killing than their untransformed progenitors. The cytotoxic effect of H-1 virus was more severe than that of MVMp. Moreover, the level of viral DNA replication was much (10- to 85-fold) enhanced in the transformants compared with their untransformed parent cells. Thus, in this system, cell transformation appears to correlate with an increase in both DNA amplification and cytotoxicity of the parvoviruses. However, the accumulation of parvovirus DNA in the transformants was not always accompanied by the production of infectious virus. Like in vitro-transformed fibroblasts, a fibrosarcoma-derived cell line was sensitive to the killing effect of both H-1 virus and MVMp and amplified viral DNA to high extents. The results indicate that oncogenic transformation can be included among cellular states which modulate permissiveness to parvoviruses under defined growth conditions.
EIa-mediated stimulation of the adenovirus EIII promoter involves an enhancer element within the nearby EIIa promoter
Authors: Zajchowski D A , Jalinot P , Kedinger C
Published: January 2020

The transcriptional induction of adenovirus early genes by the viral immediate early gene EIa constitutes an attractive model system for the study of control mechanisms involved in eucaryotic promoter function. The EIa-mediated activation of the divergently transcribed EIIa early (EIIaE) and EIII promoters was investigated in experiments in which recombinant plasmids containing the entire EIIa-EIII control region were cotransfected with a plasmid expressing the EIa 13S mRNA. First, both promoters were activated by low levels of EIa, but the extent of EIII induction decreased with increasing EIa concentrations, whereas EIIaE stimulation remained unchanged. Second, transcriptional analysis of deletion mutants revealed that an element of the EIIaE promoter contributed to maximal EIa responsiveness of the nearby EIII promoter. This element, located between positions -82 and -71 with respect to the EIIaE major cap site, corresponded to the central portion of an EIa-dependent enhancer, originally mapped between about -110 and -50 (P. Jalinot and C. Kédinger, Nucleic Acids Res. 14:2651-2669, 1986). The implication of these observations in the coordinate expression from the EIIaE and EIII promoters during lytic infection is discussed.
Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies
Authors: Taniguchi K , Hoshino Y , Nishikawa K , Green K Y , Maloy W L , Morita Y , Urasawa S , Kapikian A Z , Chanock R M , Gorziglia M
Published: January 2020

The neutralization epitopes of human and simian rotavirus protein VP7 were studied by producing six neutralizing monoclonal antibodies (N-MAbs) and using these N-MAbs to select antigenic mutants that resisted neutralization by the N-MAbs used for their selection. Cross-neutralization tests between the N-MAbs and the antibody-selected antigenic mutants identified one cross-reactive and five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site. In addition, the amino acid substitutions in human rotavirus VP7 that are responsible for the antigenic alterations in the mutants selected with anti-VP7 cross-reactive or serotype-specific N-MAbs were identified. All the amino acid substitutions in the antigenic mutants occurred in one of two variable regions: amino acids 87 to 101 and 208 to 221.
Activation of thermosensitive RNA splicing and production of a heat-labile P85gag-mos kinase by the introduction of a specific deletion in murine sarcoma virus-124 DNA
Authors: de Mars M , Cizdziel P E , Murphy E C
Published: January 2020

Murine sarcoma virus ts110 (MuSVts110) is a conditionally transformation-defective MuSV mutant lacking 1,487 bases found in its wild-type parent, MuSV-349 (MuSV-124). Expression of the MuSVts110 v-mos gene product, P85gag-mos, requires splicing of the viral transcript to align the gag and mos genes in frame. However, this splice event is restricted to growth temperatures of 33 degrees C or lower. No splicing of the viral RNA, no production of P85gag-mos, and, hence, no cell transformation is observed at growth temperatures above 33 degrees C. To determine whether thermosensitive splicing is an intrinsic property of To determine whether thermosensitive splicing is an intrinsic property of MuSVts110 RNA specified by the 1,487-base deletion or a result of a cellular defect, we examined an "equivalent" or MuSVts110 DNA (designated ts32 DNA) constructed by combining wild-type MuSV-124 DNA fragments with a synthetic oligonucleotide to yield an otherwise wild-type viral DNA containing the same 1,487-base deletion as authentic MuSVts110. As observed in control cells (6m2 cells) infected with the authentic MuSVts110 virus, NIH 3T3 cells transfected with ts32 DNA appeared morphologically transformed when grown at 33 degrees C, but were converted to a more normal, flattened shape within a few hours of a shift to 39 degrees C. In concert with these morphological changes, both the processing of the ts32 RNA transcripts and the production of ts32 p85gag-mos kinase were found to be optimal at growth temperatures from 28 to 33 degrees C, but dramatically reduced at 37 to 41 degrees C. Like authentic P85gag-mos, the ts32 P85gag-mos kinase activity was rapidly inactivated by brief exposure to 39 degrees C. These results suggested that the MuSVts110 equivalent is functionally indistinguishable from authentic MuSVts110 and that the novel temperature-sensitive splicing of MuSVts110 transcripts is specified by an intrinsic property of the viral RNA.
Sialic acids on the surface of caprine arthritis-encephalitis virus define the biological properties of the virus
Authors: Huso D L , Narayan O , Hart G W
Published: January 2020

The lentivirus caprine arthritis-encephalitis virus (CAEV) is a pathogen of goats. It is transmitted in milk and causes a persistent infection in goats, which often fail to produce neutralizing antibodies to the virus. Native CAEV particles are remarkably resistant to digestion with proteinase K and are neutralized extremely slowly by immune sera. Our studies showed that the virus particles are heavily sialylated. Studies with highly specific sialyltransferase enzymes identified penultimate carbohydrate linkages typical of O- and N-linked oligosaccharides on the virus and suggested that the virus may be more heavily sialylated on O-linked than on N-linked oligosaccharides. Removal of sialic acids from the virus by neuraminidase treatment did not reduce infectivity of the particles. However, desialylation rendered the virus more susceptible to proteolysis by proteinase K. Desialylation also enhanced the kinetics of neutralization of the virus by goat antibodies. These results suggest that the carbohydrates on the viral surface are important both in protecting viral proteins from digestion by proteases and in protecting the virus from rapid neutralization by antibodies.
Coevolution of cells and viruses in a persistent infection of foot-and-mouth disease virus in cell culture
Authors: de la Torre J C , Martínez-Salas E , Diez J , Villaverde A , Gebauer F , Rocha E , Dávila M , Domingo E
Published: January 2020

Virus and cells evolve during serial passage of cloned BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV). These carrier cells, termed C1-BHK-Rc1 (J.C. de la Torre, M. Dávila, F. Sobrino, J. Ortín, and E. Domingo, Virology 145:24-35, 1985), become constitutively resistant to the parental FMDV C-S8c1. Curing of late-passage C1-BHK-Rc1 cells of FMDV by ribavirin treatment (J.C. de la Torre, B. Alarcón, E. Martínez-Salas, L. Carrasco, and E. Domingo, J. Virol. 61:233-235, 1987) did not restore sensitivity to FMDV C-S8c1. The resistance of C1-BHK-Rc1 cells to FMDV C-S8c1 was not due to an impairment of attachment, penetration, or uncoating of the particles but to some intracellular block that resulted in a 100-fold decrease in the amount of FMDV RNA in the infected cells. FMDV R59, the virus isolated from late-passage carrier cells, partly overcame the cellular block and was more cytolytic than FMDV C-S8c1 for BHK-21 cells. Sequencing of the VP1 gene from nine viral clones from C1-BHK-Rc1 cells showed genetic heterogeneity of 5 X 10(-4) substitutions per nucleotide. Mutations were sequentially fixed during persistence. In addition to resistance to FMDV C-S8c1, C1-BHK-Rc1 cells showed a characteristic round cell morphology, and compared with BHK-21 cells, they grew faster in liquid culture, were less subject to contact inhibition of growth, and had an increased ability to form colonies in semisolid agar. Reconstitution of a persistent infection was readily attained with late-passage C1-BHK-Rc1 cells and FMDV C-S8c1 or FMDV R59. The results suggest that coevolution of BHK-21 cells and FMDV contributes to the maintenance of persistence in cell culture.
Avian retrovirus pp32 DNA endonuclease is phosphorylated on Ser in the carboxyl-terminal region
Authors: Horton R , Mumm S , Grandgenett D P
Published: January 2020

The avian retrovirus pp32 DNA endonuclease and the beta polypeptide of the reverse transcriptase contain the same three phosphoserine (p-Ser) tryptic peptides. At least 95% of the Pi label is nearly equally distributed between two major p-Ser tryptic peptides derived from either beta or pp32. These polymerase gene-derived proteins were metabolically labeled with various radioactive amino acids or Pi, and the purified protein was subjected to cyanogen bromide or hydroxylamine cleavage. The results indicated that the two major p-Ser tryptic peptides map to the COOH-termini of both proteins. The two major p-Ser tryptic peptides isolated from Pi-labeled pp32 were subjected to proteolysis by three separate specific proteases. Analysis of the data suggested that these p-Ser are located on pp32 at amino acid positions 262 and 282 from the amino terminus of pp32 (286 amino acids in length). At present, we cannot exclude the possibility that one or both p-Ser peptides map between amino acid positions 124 to 150. The role of this site-specific phosphorylation of pp32 and beta is also discussed.
Role of VP3 in human rotavirus internalization after target cell attachment via VP7
Authors: Fukuhara N , Yoshie O , Kitaoka S , Konno T
Published: January 2020

A cell lysate prepared from MA104 cells that had been infected with human rotavirus KUN strain (HRV-KUN) contained a 35-kilodalton protein capable of binding to MA104 cells. The binding of the 35-kilodalton protein was inhibited by a serotype 2-specific antiserum but not by antisera to other serotypes. Not only trypsin-treated, infectious HRV-KUN but also untreated, noninfectious virions effectively competed with the 35-kilodalton protein for the same cell surface binding sites. One monoclonal anti-VP7 (AH6) absorbed the 35-kilodalton protein from the HRV-KUN-infected cell lysate, whereas another monoclonal anti-VP7 (S2-2G10) inhibited the virions to compete with the 35-kilodalton protein for the cell surface binding sites. Both anti-VP7 (S2-2G10) and anti-VP3 (K-1532, K-376) monoclonal antibodies had the virus-neutralization activity, but only anti-VP7 inhibited virus adsorption. On the other hand, anti-VP3 monoclonal antibodies were capable of completely inhibiting the infection of preadsorbed HRV-KUN as long as virions were not yet internalized. Subsequent studies with [35S]methionine-labeled and purified HRV-KUN showed that not only trypsin-treated, infectious virions but also untreated, noninfectious virions were capable of efficient target cell binding and internalization. The internalization modes of these two HRV-KUN preparations were, however, quite different. Only the components of the inner capsid were internalized from trypsin-treated virions, whereas no such selective internalization was seen with untreated virions. Furthermore, anti-VP3 inhibited this selective internalization of the inner capsid from the infectious virions. From these results we conclude that VP7 is the HRV-KUN cell attachment protein and that adsorption of HRV-KUN via VP7 is independent of trypsin treatment, whereas the limited cleavage of VP3 by trypsin, which is essential for the development of HRV-KUN infectivity, is needed for the selective internalization of the inner capsid components, a process that is apparently essential for HRV-KUN infection.
Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity
Authors: Chang M F , Baker S C , Soe L H , Kamahora T , Keck J G , Makino S , Govindarajan S , Lai M M
Published: January 2020

The genetic origin, structure, and biochemical properties of the delta antigen (HDAg) of a human hepatitis delta virus (HDV) were investigated. A cDNA fragment containing the open reading frame encoding the HDAg was transcribed into RNA and used for in vitro translation in rabbit reticulocyte lysates. The HDAg open reading frame was also inserted into an expression vector containing a simian virus 40 T-antigen promoter and expressed into COS 7 cells. In both systems, a protein species of 26 kilodaltons was synthesized from this open reading frame and could be specifically immunoprecipitated with antisera obtained from patients with delta hepatitis. A similar protein was also synthesized from antigenomic-sense monomeric HDV RNA in both systems, although the efficiency of translation was lower than that of the isolated open reading frame. This protein was found to be phosphorylated at the serine residues. Immunoperoxidase studies with anti-HDV sera demonstrated that the HDAg was expressed mainly in the nuclei of the transfected COS 7 cells. Moreover, the HDAg was shown to bind the genomic RNA of HDV. These studies indicate that HDAg is encoded by the antigenomic-sense RNA of HDV and is a nuclear phosphoprotein associated with an RNA-binding activity.
Identification of a neutralizing epitope shared by bluetongue virus serotypes 2 and 13
Authors: Ristow S , Leendersten L , Gorham J , Yilma T
Published: January 2020

The serotypes of bluetongue virus (BTV) are classified by differences in neutralization commonly induced by P2, a major surface protein. A BTV serotype 13 (BTV-13) monoclonal antibody, 4B13-207A, immunoprecipitated P2s of BTV-13 and BTV-2 and also neutralized both viruses. These data indicate that P2s from BTV-13 and BTV-2 share a common neutralizing epitope that is not detected by neutralizing polyclonal antibody to BTV-13.
Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage
Authors: Sharmeen L , Kuo M Y , Dinter-Gottlieb G , Taylor J
Published: January 2020

The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the HDV genome. Because the site is located almost adjacent to the 3' end of the delta antigen-coding region, the only known functional open reading frame of HDV, we suggest that the cleavage may have a role not only in genome replication but also in RNA processing, helping to produce a functional mRNA for the translation of delta antigen.
Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions
Authors: Sells M A , Zelent A Z , Shvartsman M , Acs G
Published: January 2020

Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.-L. Chen, and G. Acs, Proc. Natl. Acad. Sci. USA 84:1005-1009, 1987) which elicit acute hepatitis in chimpanzees (G. Acs, M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper, Proc. Natl. Acad. Sci. USA 84:4641-4644, 1987). We report here the initial characterization of the viral nucleic acids produced in this culture system. Kinetic analyses of nuclear, cytoplasmic, and extracellular HBV DNAs were performed by Southern blotting with radiolabeled HBV strand-specific probes. The results from these analyses indicate that at the stationary cellular growth phase, there is a dramatic increase in the rate at which HBV DNA accumulates. Incomplete double- and single-stranded forms of the HBV genome were detected in the nuclear and cytoplasmic fractions as well as in the extracellular medium. In addition, the nuclear DNA apparently includes multiple complete copies of the HBV genome chromosomally integrated and full-length covalently closed circular HBV DNA. Multiple HBV-specific polyadenylated RNAs with lengths of 3.5, 2.5, and 2.1 kilobases were identified by Northern (RNA) blot analysis. S1 nuclease mapping and primer extension identified a single 3' end and multiple unique initiation sites corresponding to nucleotides just 5' to the pre-S1 region, as well as upstream and within the pre-S2 and precore regions. The nucleic acid profile obtained from these analyses is essentially a facsimile of that obtained by studying liver tissue from HBV-infected individuals.
Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine
Authors: Calain P , Roux L
Published: January 2020

By starting from a thrice-purified wild-type measles virus plaque, the generation of detectable subgenomic RNAs was achieved within a series of five serial infections of Vero cells. The evolution of these subgenomic RNAs was followed for seven serial passages and ended with the preparation of a highly interfering viral stock. On the other hand, the detection of discrete subgenomic RNAs was achieved during the first infection of Vero cells with at least one of three measles virus vaccine preparations tested. These subgenomic RNAs, which interfered very efficiently with the replication of the endogenous standard genomes upon vaccine infection but showed a moderate interfering activity with a standard virus stock derived by plaque purification from the vaccine preparation, resulted from the presence of defective interfering particles in the vaccine preparation. The relevance of this finding for the attenuation, stability, and potential capacity for persistent infection of such a vaccine is discussed.
Interaction between the octamer-binding protein nuclear factor III and the adenovirus origin of DNA replication
Authors: Pruijn G J , van Miltenburg R T , Claessens J A , van der Vliet P C
Published: January 2020

Nuclear factor III (NFIII) is a HeLa sequence-specific DNA-binding protein that stimulates initiation of adenovirus DNA replication in vitro and may be involved in regulation of transcription of several cellular and viral genes. We have studied the interaction between NFIII and the binding site in the adenovirus type 2 (Ad2) origin in detail by methidiumpropyl-EDTA.iron(II) and hydroxyl radical footprinting and by alkylation interference experiments. Our results indicate that (i) the core of the recognition sequence is 5'-TATGATAAT-3'; (ii) both major and minor groove base contacts are detected, and all base pairs in the core are involved in binding; (iii) many backbone contacts are observed divided into a large domain coinciding with the core and a small domain; (iv) contact points are not confined to one side of the DNA helix in contrast to the nuclear factor I (NFI)-binding site; (v) the binding site overlaps the NFI-binding site for at least one nucleotide. A number of Ad2 mutants as well as related binding sites in the origins of other adenovirus serotypes were systematically compared for binding with NFIII. The results are in good agreement with the contact point studies and show that at least one AT base pair is commonly required by NFI and NFIII for optimal binding. The strongest binding site, which contains the octamer/decanucleotide motif (ATGCAAAT[NA]), was found in the Ad4 origin, which lacks an NFI-binding site. Stimulation of in vitro DNA replication of Ad2, Ad4, and Ad12 by NFIII showed that the maximal level of stimulation is dependent on the affinity of NFIII for the origin.
Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene
Authors: Chisholm G E , Henner D J
Published: January 2020

The immediate-early IE-1 gene of Autographa californica nuclear polyhedrosis virus was cloned, and its nucleotide sequence was determined. Sequence analysis indicated that this gene would encode a protein of 582 amino acids with a predicted molecular weight of 66,822. Analysis of IE-1 gene expression during baculovirus infection identified two transcripts. One, 1.9 kilobases (kb), was expressed at constant steady-state levels throughout infection, whereas the other, 2.1 kb, was expressed only early in infection. Analysis of IE-1 cDNA clones demonstrated that the 2.1-kb transcript contained the entire 1.9-kb transcript (exon 1) plus an additional 5' end (exon 0). Genomic Southern analysis placed the exon 0 sequences on the EcoRI B fragment, 4 kilobase pairs upstream of exon 1. Sequencing of the upstream region identified an open reading frame whose 5' end was identical to the exon 0 sequences in the cDNAs. Examination of the genomic DNA sequences around the exon-exon junction revealed sequences similar to published consensus splice acceptor and donor sequences. This is the first example of splicing of any viral transcript during baculovirus infection.
Gene product of region E4 of adenovirus type 5 modulates accumulation of certain viral polypeptides
Authors: Hemström C , Nordqvist K , Pettersson U , Virtanen A
Published: January 2020

An adenovirus type 5 mutant, designated H5ilE4I, was constructed in which region E4 was replaced by a cloned cDNA. The cDNA was a copy of an mRNA which exclusively contains open translational reading frames 6 and 7. The phenotype of the mutant was compared with that of the previously characterized E4 mutant H2dl808 and wild-type adenovirus 5. Although the H5ilE4I mutant lacked at least five E4 genes, it was nondefective for growth in HeLa cells. The defects in viral DNA replication, late protein synthesis, and shutoff of host cell protein synthesis associated with the phenotype of the H2dl808 mutant were not observed in HeLa cells infected with the H5ilE4I mutant. However, differences were observed regarding the time of onset of viral DNA replication and the accumulation of the hexon polypeptide as well as the 72-kilodalton adenovirus-specific DNA-binding protein. The results thus indicate that open reading frame 6 or 7 or both contain all genetic information required for viral replication in tissue culture cells, whereas another E4 gene modulates the accumulation of certain viral polypeptides. The early onset of viral DNA replication in H5ilE4I-infected cells may be an indirect effect of the enhanced expression of the 72-kilodalton DNA-binding protein.
Expression of herpes simplex virus type 1 (HSV-1) latency-associated transcripts and transcripts affected by the deletion in avirulent mutant HFEM: evidence for a new class of HSV-1 genes
Authors: Spivack J G , Fraser N W
Published: January 2020

During latent herpes simplex virus type 1 (HSV-1) infection in the trigeminal ganglia of mice, three virus-specific transcripts, 2.0, 1.5, and 1.45 kilobases (kb), are detectable by Northern (RNA) blot analysis, but only the 2.0-kb transcript can be detected in HSV-1-infected tissue culture cells (J.G. Spivack and N. W. Fraser, J. Virol. 61:3842-3847, 1987). Since these latency-associated genes map to a diploid region of the genome, transcription from the deletion mutant HFEM, which contains only one complete copy of these genes, was investigated to determine the effect of gene dosage. The 4.1-kb HFEM deletion is located between the alpha genes ICP0 and ICP27. ICP0 mRNA and the 2.0-kb latency-associated transcript were present at normal levels during HFEM infection, but ICP27 mRNA and 0.9- and 1.1-kb transcripts that map near the deletion were not readily detectable. The levels of expression of one or more of these genes might be an important determinant of HSV-1 virulence in animal hosts. ICP27 mRNA accumulated when protein synthesis was inhibited before HFEM infection, implying that the deletion may affect ICP27 regulatory rather than coding elements. Expression of the 2.0-kb latency-associated transcript was characterized in infected CV-1 cells with metabolic inhibitors and strand-specific probes. On the basis of metabolic inhibitor studies, the gene encoding the 2.0-kb latency-associated transcript is not an alpha gene. During HSV-1 replication in infected tissue culture cells, the beta and gamma genes require the prior expression of alpha gene products. However, the latency-associated RNAs are expressed in the absence of detectable levels of alpha transcripts in latently infected mice. Thus, this latency-associated gene family appear to be regulated quite differently than alpha, beta, or gamma genes. For these reasons, and because the latency-associated genes may perform latent rather than replicative functions, we propose that they should be considered members of a new HSV-1 gene class, the lambda genes.
Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein
Authors: Trempe J P , Carter B J
Published: January 2020

Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.
Long terminal repeat (LTR) sequences, env, and a region near the 5' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1
Authors: Brown D W , Blais B P , Robinson H L
Published: January 2020

A series of recombinants between Rous-associated virus type 0 (RAV-0), RAV-1, and a replication-competent avian leukosis virus vector (RCAN) have been tested for disease potential in day-old inoculated K28 chicks. RAV-0 is a benign virus, whereas RAV-1 and RCAN induce lymphoma and a low incidence of a variety of other neoplasms. The results of the oncogenicity tests indicate that (i) the long terminal repeat regions of RAV-1 and RCAN play a major role in disease potential, (ii) subgroup A envelope glycoproteins are associated with a two- to fourfold higher incidence of lymphoma than subgroup E glycoproteins, and (iii) certain combinations of 5' viral and env sequences cause osteopetrosis in a highly context-dependent manner. Long terminal repeat and env sequences appeared to influence lymphomogenic potential by determining the extent of bursal infection within the first 2 to 3 weeks of life. This would suggest that bursal but not postbursal stem cells are targets for avian leukosis virus-induced lymphomogenesis. The induction of neutralizing antibody had no obvious influence on the incidence of lymphoma.
An orbivirus of mosquitoes which induces CO2 sensitivity in mosquitoes and is lethal for rabbits
Authors: Vazeille M C , Rosen L , Guillon J C
Published: January 2020

An orbivirus, JKT-7400, isolated from Culex mosquitoes in Indonesia, replicated to a high titer and induced cytopathic effects in Aedes albopictus cell cultures. The virus produced lethal sensitivity to carbon dioxide in Culex and Aedes mosquitoes as well as in Drosophila melanogaster fruit flies but was not the agent of the hereditary sensitivity to carbon dioxide previously described for Culex quinquefasciatus. When injected intravenously in high doses, JKT-7400 virus was lethal for rabbits, apparently without replicating to a significant extent. It was not pathogenic for adult mice inoculated intravenously or for adult or suckling mice inoculated intracerebrally and intraperitoneally. Unlike an orbivirus isolated from Culex mosquitoes in China, JKT-7400 did not interfere with the replication of Japanese encephalitis virus in mosquitoes.
Identification of a novel retroviral gene unique to human immunodeficiency virus type 2 and simian immunodeficiency virus SIVMAC
Authors: Kappes J C , Morrow C D , Lee S W , Jameson B A , Kent S B , Hood L E , Shaw G M , Hahn B H
Published: January 2020

Human and simian immunodeficiency-associated retroviruses are extraordinarily complex, containing at least five genes, tat, art, sor, R, and 3' orf, in addition to the structural genes gag, pol, and env. Recently, nucleotide sequence analysis of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus SIVMAC revealed the existence of still another open reading frame, termed X, which is highly conserved between these two viruses but absent from HIV-1. In this report, we demonstrate for the first time that the X open reading frame represents a functional retroviral gene in both HIV-2 and SIVMAC and that it encodes a virion-associated protein of 14 and 12 kilodaltons, respectively. We also describe the production of recombinant TrpE/X fusion proteins in Escherichia coli and show that sera from some HIV-2-infected individuals specifically recognize these proteins.
Trypsin-sensitive neutralization site on VP1 of Theiler's murine encephalomyelitis viruses
Authors: Ohara Y , Senkowski A , Fu J L , Klaman L , Goodall J , Toth M , Roos R P
Published: January 2020

We generated Theiler's murine encephalomyelitis virus mutants resistant to several neutralizing monoclonal antibodies (MAbs) having their epitopes near a trypsin cleavage site of VP1. Neutralization and Western blot (immunoblot) studies suggest that two of the MAbs have identical epitopes that partly overlap the epitope of a third MAb. Sequencing of RNA of the mutants localized the epitopes to a site near the carboxyl end of VP1. The limited diversity of nucleotide changes seen in the mutants and the immunodominance of the site suggest that the carboxyl end of VP1 may have an important function.
Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus
Authors: Dewar R L , Natarajan V , Vasudevachari M B , Salzman N P
Published: January 2020

A recombinant adenovirus was constructed by inserting the human immunodeficiency virus type 1 (HIV-1) envelope gene downstream from the early region 3 (E3) promoter of adenovirus type 5 (Ad5), replacing the coding sequences of E3. The recombinant virus replicated as efficiently as the parent virus in all cell lines tested. Human cells infected with the recombinant virus synthesized the HIV-1 envelope precursor gp160, which was efficiently processed to the envelope glycoproteins gp120 and gp41. A human T-lymphoblast line (Molt-4) infected with the recombinant virus expressed HIV-1 envelope glycoproteins on the cell surface, leading to syncytium formation. The envelope gene was expressed from the E3 promoter at early times after infection and at late times from the major late promoter. When cotton rats were infected with the recombinant virus, antibodies against the HIV-1 envelope glycoproteins could be expressed in an immunoreactive form by the recombinant adenovirus, further illustrating the usefulness of adenoviruses as expression vectors.