bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of asymptomatic herpes simplex virus infections after vaccination
Authors: Ashley R , Mertz G J , Corey L
Published: January 2020

Twenty-two volunteers seronegative for antibodies to herpes simplex virus (HSV) were enrolled in a trial to determine tolerance and immunogenicity of an HSV-2 glycoprotein subunit vaccine. Vaccine was administered at days 0, 28, and 140, and sera were obtained on days 0, 7, 14, 21, 28, 35, 49, 56, 140, 147, and 365 for determination of HSV neutralizing antibody activity and antibody-dependent cell cytotoxicity (ADCC). Sera were also tested by immunoprecipitation of radiolabeled HSV-2-infected cell proteins and polyacrylamide gel electrophoresis to identify the viral proteins which elicited antibody responses in vaccine recipients. After vaccination two male volunteers presented with atypical first-episode genital herpes: patient 1 with a culture-negative genital lesion at day 53 and patient 3 with urethritis at day 68. Seroconversion to wild-type viral proteins not present in the vaccine was detectable by radioimmunoprecipitation-polyacrylamide gel electrophoresis within 10 days in both patients. Two additional volunteers, one a sex contact of patient 1, seroconverted asymptomatically to nonvaccine proteins during the trial. All four vaccine breakthrough patients were indistinguishable from the other volunteers in the time required to develop neutralizing and ADCC antibodies, in the titer of these antibodies, and the time to seroconversion to gB and gD vaccine proteins. However, only one of the four breakthrough patients had antibodies to g80 (a complex of gC-2 and gE) after vaccination as compared with 15 of the other 18 volunteers (P = 0.05). Neither neutralizing antibody nor ADCC titers consistently identified acquisition of wild-type viral infection; therefore, protein-specific serologies were required to detect wild-type antibodies in these four patients. These data underscore the importance of using serologic assays which will distinguish naturally acquired infection from the immune response to vaccination.
Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites
Authors: Rohdewohld H , Weiher H , Reik W , Jaenisch R , Breindl M
Published: January 2020

The chromatin conformation of mouse genome regions containing Moloney murine leukemia proviral intergration sites in two Mov mouse strains and randomly selected integration sites in virus-infected mouse 3T3 fibroblasts was analyzed. All integrations have occurred into chromosomal regions containing several DNase-hypersensitive sites, and invariably the proviral integration sites map within a few hundred base pairs of a DNase-hypersensitive site. The probability that this close association between proviral integration sites and DNase-hypersensitive sites was due to chance was calculated to be extremely low (2 X 10(-4]. Because the proviral integrations analyzed were not selected for an altered phenotype, our results suggest that DNase-hypersensitive regions are preferred targets for retrovirus integration.
Identification of two new bacteriophage T4 genes that may have roles in transcription and DNA replication
Authors: Hsu T , Wei R X , Dawson M , Karam J D
Published: January 2020

We have identified two bacteriophage T4 genes, 45.1 and 45.2, that map in the intergenic space between phage replication genes 46 (which encodes a recombination initiation protein) and 45 (which encodes a bifunctional protein required in replication and transcription). The existence of genes 45.1 and 45.2 had not been previously recognized by mutation analysis of the T4 genome. We cloned the T4 gene 45.1/45.2 segment, determined its nucleotide sequence, and expressed its two reading frames at high levels in bacterial plasmids. The results predicted molecular weights of 11,400 (100 amino acids) for gp45.1 and 7,500 (62 amino acids) for gp45.2. We also determined that in T4-infected Escherichia coli, genes 45.1 and 45.2 are cotranscribed with their distal neighbor, gene 45, by at least one mode of transcription. In an accompanying report (K. P. Williams, G. A. Kassavetis, F. S. Esch, and E. P. Geiduschek, J. Virol. 61:600-603, 1987), it is shown that the product of gene 45.1 is the so-called T4-induced 15K protein, an RNA polymerase-binding protein of unknown role in phage development. Possibly, T4 genes 45.2, 45.1, and 45 constitute an operon for host RNA polymerase-binding phage proteins. Jointly with Williams et al., we propose the term rpb (RNA polymerase-binding) to refer to T4 genes whose products bind to the host RNA polymerase and have adopted the name rpbA for T4 gene 45.1.
Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein
Authors: Walter G , Carbone A , Welch W J
Published: January 2020

Affinity-purified medium T antigen encoded by NG59, a nontransforming mutant of polyomavirus, is specifically associated with a protein of 72,000 daltons (72K protein). Medium T antigens of wild-type polyomavirus and the transformation-competent mutant dl8 are not associated with the 72K protein. Instead, they form a complex with another protein of 61,000 daltons. Several lines of evidence suggest that the medium T antigen-associated 72K protein is equivalent to the abundant and constitutive 73K heat shock protein. First, on two-dimensional polyacrylamide gels the 72K protein migrated with the same pI (5.6) as did the 73K heat shock protein. Second, the 72K protein was immunoprecipitable with antibodies against heat shock proteins. Third, when digested with V8 protease, the 72K protein gave rise to the same pattern of fragments as did the 73K heat shock protein.
Epstein-Barr virus glycoprotein homologous to herpes simplex virus gB
Authors: Gong M , Ooka T , Matsuo T , Kieff E
Published: January 2020

The Epstein-Barr virus DNA open reading frame BALF4 (R. Baer, A.T. Bankier, M.D. Biggin, P.L. Deininger, P.J. Farrell, T.J. Gibson, G. Hatfull, G.S. Hudson, S.C. Stachwell, C. Sequin, P.S. Tuffnell, and B.G. Barrell, Nature [London] 310:207-211, 1984), which by nucleotide sequence comparison could encode a protein similar to herpes simplex virus gB (P.E. Pellett, M.D. Biggin, B. Barrell, and B. Roizman, J. Virol. 56:807-813, 1985), has now been shown to encode a 110-kilodalton glycoprotein. Late infectious cycle RNAs of 3.0 and 1.8 kilobases are transcribed from BALF4. Translation of these RNAs in vitro, transcription and translation of BALF4 in vitro, or metabolic labeling of cells in the presence of tunicamycin and immunoprecipitation with BALF4-specific sera results in identification of a 93-kilodalton precursor to gp110. Since N-glycosidase F only reduces the size of gp110 to 105 kilodaltons, gp110 probably has both N- and O-linked glycosylation, gp110 is an abundant glycoprotein in Epstein-Barr virus-infected cells. In infected lymphocytes and in 3T3 cells, in which the gene is expressed from a recombinant expression vector, most of the protein is cytoplasmic and perinuclear. In contrast to gB, gp110 was not detected in the infected-cell plasma membrane. In cells replicating Epstein-Barr virus, gp110 localized to the inner and outer nuclear membrane lamellae and to endoplasmic reticulum structures which sometimes contained enveloped virus. gp110 may play an important role in modifying infected intracellular membranes.
Organization and nucleotide sequence of a densovirus genome imply a host-dependent evolution of the parvoviruses
Authors: Bando H , Kusuda J , Gojobori T , Maruyama T , Kawase S
Published: January 2020

The genome structure of a densovirus from a silkworm was determined by sequencing more than 85% of the complete genome DNA. This is the first report of the genome organization of an insect parvovirus deduced from the DNA sequence. In the viral genome, two large open reading frames designated 1 and 2 and one smaller open reading frame designated 3 were identified. The first two open reading frames shared the same strand, while the third was found in the complementary sequence. Computer analysis suggested that open reading frame 2 may encode all four structural proteins. The genome organization and a part of the nucleotide sequence were conserved among the insect densovirus, rodent parvoviruses, and a human dependovirus. These viruses may have diverged from a common ancestor.
Herpes simplex virus type 1 glycoprotein E is not indispensable for viral infectivity
Authors: Neidhardt H , Schröder C H , Kaerner H C
Published: January 2020

A mutant of the herpes simplex virus type 1 Angelotti was isolated in which 87% of the coding region of glycoprotein E (gE) was deleted and replaced by a functional neomycin resistance gene of the Tn5 transposon. The mutant was characterized by restriction enzyme analyses and Southern blotting. Western blotting of proteins and immunofluorescence assays revealed that gE was completely absent and that the Fc receptor was not expressed in cells infected with the mutant. The fact that this mutant was viable and that it replicated to a slightly lower titer than did the wild-type virus suggests that the presence of gE is not a prerequisite of viral infectivity in tissue culture.
A nonstructural protein of feline panleukopenia virus: expression in Escherichia coli and detection of multiple forms in infected cells
Authors: Carlson J O , Lynde-Maas M K , Shen Z D
Published: January 2020

Sequences coding for the nonstructural protein NS1 of the autonomous parvovirus feline panleukopenia virus were expressed in Escherichia coli as fusion proteins. The fusion proteins were specifically bound by antisera from canine parvovirus-infected dogs. Antisera against one of the fusion proteins bound to several proteins found only in feline panleukopenia virus-infected feline cells.
Location, transcription, and sequence of a baculovirus gene encoding a small arginine-rich polypeptide
Authors: Wilson M E , Mainprize T H , Friesen P D , Miller L K
Published: January 2020

The nucleotide sequence of the cDNA of an abundant late 0.5-kilobase transcript of Autographa californica nuclear polyhedrosis virus revealed a small open reading frame encoding an arginine-rich 6.9-kilodalton protein. The predicted amino acid composition of the 6.9-kilodalton protein was essentially identical to that of the core protein of viral nucleocapsids. The precise location of the 5' and 3' ends of the transcript were confirmed by S1 nuclease and primer extension analyses. Multiple overlapping transcripts through this region include three early and three abundant late RNAs which are transcribed counterclockwise and one transient RNA which is transcribed clockwise with respect to the physical map of the virus.
Disease specificity of nondefective Friend and Moloney murine leukemia viruses is controlled by a small number of nucleotides
Authors: Li Y , Golemis E , Hartley J W , Hopkins N
Published: January 2020

Moloney murine leukemia virus induces T cell lymphomas after injection into NFS mice, whereas the nondefective Friend virus induces erythroleukemias. Previous studies showed that sequences encompassing the viral transcriptional signals in U3 are the primary determinant of this phenotype in recombinants between these two viruses. To more precisely identify the sequences responsible, we constructed additional recombinants, within U3, between Friend and Moloney viruses and assayed these recombinants for for their disease specificity. We found that a fragment 191 bases long that included the direct repeat (enhancer) region plus 22 nucleotides to its 3' side from Friend virus was sufficient to convert Moloney virus to a virus that induced only erythroleukemias. A 171-base-long fragment of Moloney virus, including just the direct repeat, converted Friend virus to a virus that induced primarily lymphomas (about 85% of mice injected). We also constructed Moloney and Friend virus variants with one rather than two copies of the enhancer element. These viruses retained their disease specificity, although they exhibited a marked increase in the latent period of disease induction. Together the results suggest that 25 or fewer nucleotide differences, lying within and also just 3' of the direct repeat, are the primary determinant of the distinct disease specificities of nondefective Friend and Moloney viruses.
Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection
Authors: Crittenden L B , McMahon S , Halpern M S , Fadly A M
Published: January 2020

We inoculated susceptible chicken embryos with the endogenous avian leukosis virus Rous-associated virus-0 (RAV-0) on day 6 of incubation. At 1 week after hatching, RAV-0-infected and control chickens were inoculated with either RAV-1 or RAV-2, exogenous viruses belonging to subgroups A and B, respectively. The chickens injected with RAV-0 as embryos remained viremic with exogenous virus longer and either failed to develop type-specific humoral immunity to exogenous virus or developed it later than the control chickens not inoculated with RAV-0. The RAV-0-injected chickens also developed neoplasms at a much higher frequency than did the control chickens. We suggest that the lower immune responses of the RAV-0-injected chickens were due to an immunological tolerance to envelope group-specific glycoproteins shared among endogenous and exogenous viruses.
Macrophages from endotoxin-hyporesponsive (Lpsd) C3H/HeJ mice are permissive for vesicular stomatitis virus because of reduced levels of endogenous interferon: possible mechanism for natural resistance to virus infection
Authors: Vogel S N , Fertsch D
Published: January 2020

The C3H/HeJ mouse strain bears an autosomal gene defect, Lpsd, which results in a greatly diminished capacity to respond to endotoxin, the ubiquitous lipopolysaccharide derived from the cell walls of gram-negative bacteria. These mice also exhibit greater susceptibility to a variety of viral and bacterial infections than syngeneic, fully lipopolysaccharide-responsive (Lpsn) mouse strains and possess macrophages with defects in differentiation which are reversed by treatment with exogenous interferon (IFN). To test directly the hypothesis that C3H/HeJ macrophages are deficient in endogenous IFN levels, macrophages from C3H/HeJ (Lpsd) and C3H/OuJ (Lpsn) mice were compared for sensitivity to vesicular stomatitis virus. At a multiplicity of infection of 0.1, C3H/OuJ macrophages were completely refractory to infection, whereas C3H/HeJ macrophages were permissive for replication, and infection resulted in 100% cytopathic effect. These findings were confirmed with a second inbred Lpsn and Lpsd strain pair. Levels of 2',5'-oligoadenylate synthetase were significantly higher in Lpsn cells. C3H/HeJ macrophages, derived from bone marrow precursors under the influence of macrophage colony-stimulating factor, shown previously to induce IFN in macrophages, were as refractory as C3H/OuJ macrophages. Exposure of nonpermissive macrophages to anti-IFN-alpha/beta antibody prior to infection rendered cells permissive. Our findings suggest that endotoxin provides a primary stimulus for the maintenance of normal macrophage differentiation and innate resistance via the induction of endogenous IFN by macrophages.
Resolution of a polyomavirus-mouse hybrid replicon: viral function required for recombination
Authors: Piché A , Bourgaux P
Published: January 2020

RmI, a circular chimera made of the polyomavirus (Py) genome with an insertion of mouse DNA (Ins), effectively undergoes intramolecular recombination in normal mouse cells, as indicated by the conversion of cloned RmI (RmIc) into unit-length Py DNA in transfected cultures. To follow the fate of the cellular component of RmI after recombination, the origin of simian virus 40 (SV40) DNA was inserted into the Ins region of RmIc, generating a new molecular species designated SV-RmIc. The recombination of SV-RmIc in simian cells synthesizing SV40 large T antigen gave rise to a molecule containing the SV40 origin, the reciprocal of unit-length Py DNA. However, SV-RmIc failed to yield unit-length Py DNA in murine cells unless Py large T antigen was provided in trans. In murine cells synthesizing SV40 large T antigen, the only detectable product from SV-RmIc contained only Py sequences, but was heterogeneous in size. These results and others also reported here strongly suggest that Py large T antigen plays a direct role in the resolution of RmI in murine cells.
Posttranslational processing of an Epstein-Barr virus-encoded membrane protein expressed in cells transformed by Epstein-Barr virus
Authors: Baichwal V R , Sugden B
Published: January 2020

The BamHI Nhet fragment of the B958 strain of Epstein-Barr virus (EBV) encodes a membrane protein (BNLF-1) that is present in cells transformed by EBV. We made a hybrid protein in which a polypeptide sequence from the carboxyl-terminal part of BNLF-1 is fused to Escherichia coli beta-galactosidase. This hybrid protein was used to immunize rabbits, and the resulting antiserum was purified by immunoaffinity chromatography. The antiserum was able to immunoprecipitate BNLF-1 from cell lysates. We found that BNLF-1 is phosphorylated at serines in EBV genome-positive B-cell lines. Pulse-chase analyses with [35S]methionine indicated that BNLF-1 is turned over in lymphoblasts with a half-life of approximately 5 h. Protein immunoblots of EBV genome-positive B-cell lines revealed both a 62,000-molecular-mass band corresponding to BNLF-1 and a myriad of lower-molecular-mass bands. We postulate that these lower-molecular-mass bands are degradation products resulting from the turnover of BNLF-1 in cells. The BNLF-1 gene was expressed in COS cells, and the protein was both phosphorylated and turned over in these cells.
Topographical rearrangements of visna virus envelope glycoprotein during antigenic drift
Authors: Stanley J , Bhaduri L M , Narayan O , Clements J E
Published: January 2020

Visna virus undergoes antigenic drift during persistent infection in sheep and thus eludes neutralizing antibodies directed against its major envelope glycoprotein, gp135. Antigenic variants contain point mutations in the 3' end of the genome, presumably within the envelope glycoprotein gene. To localize the changes in the viral proteins of antigenic mutants, we isolated 35 monoclonal antibodies (MAbs) against the envelope glycoprotein gp135 or the major core protein p27 of visna virus. The MAbs defined five partially overlapping epitopes on gp135. We used the MAbs and polyclonal immune sera directed against visna virus, gp135, or p27 in enzyme-linked immunosorbent assays to compare visna virus (strain 1514) with antigenic mutants (LV1-1 to LV1-6) previously isolated from a single sheep persistently infected with plaque-purified strain 1514. Polyclonal immune sera and anti-core p27 MAbs failed to distinguish antigenic differences among the viruses. By contrast, the anti-gp135 MAbs detected changes in all five epitopes of the envelope glycoprotein. Three gp135 epitopes, prominently exposed on strain 1514, were lost or obscured on the mutants; two covert gp135 epitopes, poorly exposed on strain 1514, were reciprocally revealed on the mutants. Even virus LV1-2, which is indistinguishable from parental strain 1514 by serum neutralization tests and which differs from it by only two unique oligonucleotides on RNase-T1 fingerprinting, displayed global changes in gp135. Our data suggest that visna virus variants may emerge more frequently during persistent infection than can be detected by serological tests involving the use of polyclonal immune sera, and the extent of phenotypic changes in their envelope glycoproteins may be greater than predicted by the small number of genetic changes previously observed. We suggest that topographical rearrangements in the three-dimensional structure of gp135 may magnify the primary amino acid sequence changes caused by point mutations in the env gene. This may complicate strategies to construct lentiviral vaccines by using the envelope glycoprotein.
Correlation of leukemogenic potential of murine retroviruses with transcriptional tissue preference of the viral long terminal repeats
Authors: Short M K , Okenquist S A , Lenz J
Published: January 2020

Recombination studies have established that retroviral long terminal repeats (LTRs) are important genetic determinants of the viral capacity to induce hematopoietic tumors and to specify the type of cell making up the tumor. Plasmids containing LTRs of several murine leukemia viruses linked to the chloramphenicol acetyltransferase gene were tested in transient assays to measure relative rates of transcriptional activity in different types of hematopoietic cells. LTRs of the thymomagenic viruses SL3-3, Moloney leukemia virus, and a Moloney mink cell focus-forming virus all expressed to higher levels than other LTRs in T-lymphocyte cell lines. Conversely, the LTRs of Friend leukemia virus and a polycythemic spleen focus-forming virus expressed to higher levels than other LTRs in erythroleukemia cells. The LTR of nonleukemogenic Akv virus induced a relatively low level of activity compared with the others in all cells tested. Thus the relative level of LTR-driven expression in various types of cells corresponds to the type of tumor caused by the intact virus in vivo. These results provide direct evidence that the tissue specificity of the transcriptional activity of LTRs plays a critical role in determining the target cell for retroviral oncogenesis.
Localization of the phosphorylations of polyomavirus large T antigen
Authors: Bockus B J , Schaffhausen B
Published: January 2020

Polyomavirus large T antigen is phosphorylated on both serine and threonine residues at a ratio of approximately 6 to 1. This phosphorylation could be resolved into a series of nine Staphylococcus aureus V8 phosphopeptides. All of these were found in an N-terminal chymotryptic fragment with a molecular weight of 57,000. A C-terminal formic acid fragment of 50,000-molecular-weight lacked phosphate. Therefore, unlike simian virus 40 large T antigen, polyomavirus large T antigen has no significant C-terminal phosphorylation. Limited V8 and hydroxylamine cleavage showed that the phosphorylations can be localized to two different portions of the molecule. A significant fraction of the phosphate was localized in the N-terminal portion of the molecule before residue 183. Within this region V8 peptides 4, 8, and 9 represented phosphorylations that were more proximal, while peptides 1, 2, and 3 included more distal phosphorylations. None of these phosphorylations appeared analogous to those of simian virus 40 large T antigen. V8 phosphopeptides 5 and 7 were more distal and could be distinguished in biological experiments from the N-terminal phosphorylations. Formic acid mapping suggested that much, if not all, of this phosphorylation is located between residues 257 and 285.
Dsi-1, a region with frequent proviral insertions in Moloney murine leukemia virus-induced rat thymomas
Authors: Vijaya S , Steffen D L , Kozak C , Robinson H L
Published: January 2020

Dsi-1 is a region of chromosomal DNA that underwent proviral insertion in 3 of 24 Moloney murine leukemia virus-induced rat thymomas. In one of these tumors, a provirus is also integrated adjacent to the proto-oncogene c-myc. The proviruses in Dsi-1 have been characterized and appear to be complete. The proviruses were located within a 2-kilobase region that contained four prominent DNase I-hypersensitive sites. These hypersensitive sites were observed in Moloney murine leukemia virus-induced thymomas but not in NRK cells. The region of Dsi-1 immediately 3' to the insertions cross-hybridized with human and chicken DNA, indicating that it contains highly conserved sequences. No evidence could be found for the expression of this highly conserved region. Dsi-1 was mapped to mouse chromosome 4. This location demonstrates that Dsi-1 is different from 16 of the known proto-oncogenes (c-abl, c-erbA c-erbB, c-ets-1, c-ets-2, c-fes, c-fos, c-myb, c-myc, c-raf, A-raf, c-Ha-ras, c-Ki-ras, N-ras, c-sis, and c-src) and 12 cellular regions of tumor-associated integrations in retrovirus-induced tumors (c-erbB, Fis-1, int-1, int-2, Mis-1/pvt-1, Mlvi-1, Mlvi-2, c-mos, c-myb, c-myc, Pim-1, and c-Ha-ras). Hybridization experiments indicated that Dsi-1 is probably different from five additional proto-oncogenes (c-fgr, c-fms, c-mos, neu, and c-yes) and from two additional frequent integration regions (lck and Mlvi-3).
In vivo phosphorylation and protein analysis of hepatitis B virus core antigen
Authors: Roossinck M J , Siddiqui A
Published: January 2020

The C open reading frame of the hepatitis B virus contains two in-frame ATG codons that are separated by the precore region and encodes two major polypeptides that are antigenically distinct and that are probably synthesized from individual mRNAs. The precore region directs the secretion of the e antigen, whereas the core antigen can be expressed in the absence of these sequences. In this report a transient expression system was used to study the hepatitis B virus core antigen. By using a chimeric complex of adenovirus major late promoter-simian virus 40 enhancer sequences, we were able to achieve high levels of core antigen expression in transfected cells, permitting characterization of this protein and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core polypeptide is a 20.9-kilodalton protein, and we show in this study that it is phosphorylated in vivo. Cell fractionation studies, the results of which are supported by indirect immunofluorescence, localized the phosphocore in the cytosol and the nucleus and indicated that it is associated with the membrane of transfected cells. Results of Triton X-114 solubilization studies indicated that the phosphocore is peripherally associated with cytoplasmic membranes. Expression of the membrane-associated phosphocore occurred in the absence of the precore sequences. The phosphocore also assembled into particles in the absence of other viral gene products or intact DNA.
Simian immunodeficiency virus induces expression of class II major histocompatibility complex structures on infected target cells in vitro
Authors: Kannagi M , Kiyotaki M , King N W , Lord C I , Letvin N L
Published: January 2020

The human immunodeficiency virus (HIV) and the closely related simian immunodeficiency virus (SIV) induce profound immune dysfunction in primate species. The present studies show that cell populations infected in vitro with SIV exhibit increases in major histocompatibility complex (MHC) class II antigen expression. Cell lines chronically infected with both the monkey and human viruses express substantially more MHC class II but not more lineage-restricted or activation antigens on their membranes than do uninfected cell lines. Furthermore, 2'-deoxy-5-iodouridine increased MHC class II antigen expression on SIV-infected cell lines in parallel with increased expression of viral antigens. MHC class II induction does not appear to be mediated through the production of a soluble factor, such as gamma interferon, by SIV-infected cells. Interestingly, studies of the kinetics of antigen expression by cell lines after SIV infection indicate that the induction of MHC class II structures is a late event. Immunoelectron microscopy revealed that MHC class II antigen is expressed not only on the surfaces of the SIV-infected cells but also on the envelope of virus particles derived from those cells. MHC antigen expression on virus-infected cells and the expression of those determinants by the virus may play a role in the pathogenesis of acquired immunodeficiency syndrome and the autoimmune abnormalities observed in HIV-infected individuals.
Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system
Authors: Estes M K , Crawford S E , Penaranda M E , Petrie B L , Burns J W , Chan W K , Ericson B , Smith G E , Summers M D
Published: January 2020

Rotaviruses are the major pathogens that cause life-threatening diarrhea in young children and animals. We inserted a simian rotavirus SA11 gene 6 cDNA into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. The major capsid antigen (VP6) was expressed in high yields (20 to 150 micrograms/10(6) cells) when Spodoptera frugiperda cells were infected with baculovirus recombinants containing SA11 gene 6 inserts. Reactivity with monospecific polyclonal and monoclonal antibodies suggested that VP6, expressed intracellularly or found in the media, maintained native antigenic determinants. VP6 purified from the media from infected cells also possessed a native oligomeric structure, was immunogenic in guinea pigs, and was able to spontaneously assemble into morphologic subunits. Antisera from immunized guinea pigs failed to neutralize virus in plaque reduction assays, but detected homologous and heterologous rotavirus strains when tested by immunofluorescence, immunoprecipitation, and enzyme-linked immunosorbent assays.
Analysis of the complete nucleotide sequence of the picornavirus Theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses
Authors: Pevear D C , Calenoff M , Rozhon E , Lipton H L
Published: January 2020

Theiler's murine encephalomyelitis viruses (TMEV) are naturally occurring enteric pathogens of mice which constitute a separate serological group within the picornavirus family. Persistent TMEV infection in mice provides a relevant experimental animal model for the human demyelinating disease multiple sclerosis. To provide information about the TMEV classification, genome organization, and protein processing map, we determined the complete nucleotide sequence of the TMEV genome and deduced the amino acid sequence of the polyprotein coding region. The RNA genome, which is typical of the picornavirus family, is 8,098 nucleotides long. The 5' untranslated region is 1,064 nucleotides long (making it the longest in the picornavirus family after the aphthoviruses) and lacks a poly(C) tract. Computer-generated comparison of the 5' and 3' noncoding regions and polyprotein revealed the highest level of nucleotide and predicted amino acid identity between the TMEV and the cardioviruses encephalomyocarditis virus (EMCV) and Mengo virus. The TMEV polyprotein, which appears to be processed like EMCV since the amino acids flanking the putative proteolytic cleavage sites have been conserved, begins with a short leader peptide followed by 11 other gene products in the standard L-4-3-4 picornavirus arrangement. Because of these similarities, we propose that the TMEV be grouped with the cardioviruses. However, since TMEV and EMCV have different biophysical properties and show no cross-neutralization, they most likely belong in a separate cardiovirus subgroup.
Expression of interferon-inducible genes in RD-114 cells
Authors: Kusari J , Tiwari R K , Kumar R , Sen G C
Published: January 2020

RD-114 is a cell line which is partially responsive to interferon (IFN). Although both IFN-alpha and IFN gamma inhibit production of the resident retrovirus, they do not inhibit replication of other viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, in these cells. In the studies reported here, we studied the characteristics of induction of seven IFN-inducible mRNAs in RD-114 cells. We observed that mRNAs 561, 6-16, 1-8, 2A, and 6-26 have similar induction characteristics in RD-114 cells and in HeLa cells, a fully responsive line. mRNA 2'-5'-oligo-adenylate synthetase (2-5(A) synthetase), however, was induced more efficiently by IFN-alpha in HeLa cells than in RD-114 cells. The same was true for the induction of metallothionein II mRNA by IFN-gamma. However, the latter mRNA was induced equally strongly in both lines when ZnCl2 was used as the inducer, suggesting that the gene is not defective in RD-114 cells. Although IFN-alpha induced 2-5(A) synthetase mRNA poorly and IFN-gamma did not induce it at all in these cells, a mixture of IFN-alpha and IFN-gamma induced this mRNA quite effectively, to a level of induction comparable to that in HeLa cells. Only 1 U of IFN-gamma per ml was sufficient to elicit this synergism, and the data suggested that an IFN-gamma-inducible protein was needed for this process. Induction of mRNA 561 by IFN-alpha in RD-114 cells, unlike that in HeLa cells, did not need ongoing protein synthesis. Once induced, this mRNA turned over rapidly in both cell lines, and this turnover could be slowed down by inhibiting protein synthesis in either cell line. IFN-induced mRNAs, such as 561 and 1-8, were polysome associated in IFN-treated RD-114 cells, suggesting that they were actively translated. Therefore, it is unlikely that the products of these IFN-inducible genes, by themselves, mediate the inhibition of replication of those viruses which are insensitive to IFN action in RD-114 cells.
Multiple tandemly repeated binding sites for cellular nuclear factor 1 that surround the major immediate-early promoters of simian and human cytomegalovirus
Authors: Jeang K T , Rawlins D R , Rosenfeld P J , Shero J H , Kelly T J , Hayward G S
Published: January 2020

We show that the large DNA genomes of human and simian cytomegaloviruses (HCMV and SCMV, respectively) each contain multiple binding sites for purified cellular nuclear factor 1 (NF1) protein. Examination of the major immediate-early (IE) gene region in the HindIII H fragment of SCMV (Colburn) by filter binding assays showed that it competed 45-fold better than the single adenovirus type 2 binding site for NF1 protein and that it contained at least two distinct binding loci. Direct DNase I footprinting analyses of the 5' upstream locus detected at least 20 adjacent NF1-binding sites located between positions -600 and -1300 relative to the IE94 mRNA start site. DNA sequence analysis of the region revealed a conserved consensus NF1 recognition element (T)TGG(C/A)N5GCCAA embedded within each of 23 highly diverged 30-base-pair tandem repeats, together with a second downstream cluster of five consensus NF1-binding sites between positions +470 and +570 in the large first intron. Two separate NF1-binding loci were also found in the equivalent IE68 gene of HCMV(Towne) DNA, but in this case the DNA sequence and competition filter binding experiments indicated a maximum of only four to five consensus binding sites encompassing the promoter-enhancer region. In transient expression assays, neither the isolated upstream IE94 tandem repeats nor a synthetic single-copy consensus NF1-binding site acted as transcriptional cis activators or enhancers when placed adjacent to the simian virus 40 minimal early region promoter. We conclude that the large and complex 5' upstream promoter-regulatory region for the SCMV IE94 gene comprises two distinct domains. The previously described four sets of 13- to 18-base-pair interspersed repeat elements between -55 and -580 provide most of the high basal transcriptional strength, whereas the arrangement of further upstream tandemly repeated NF1-binding sites may contribute significantly to the expanded biological host range for expression of SCMV IE94 compared with HCMV IE68.
Accumulation of 2',5'-oligoadenylates in encephalomyocarditis virus-infected mice
Authors: Hearl W G , Johnston M I
Published: January 2020

Levels of 2',5'-oligoadenylates (2-5A) in various tissues of murine encephalomyocarditis virus (EMCV)-infected mice were determined and compared with those found in pathogen-free mice and in mice treated with the interferon inducer poly(I).poly(C). In control, pathogen-free mice, liver, spleen, brain, and kidney tissues possessed levels of 2-5A below 1 pmol/g of tissue, demonstrating that 2-5A was not a major component of uninfected mouse tissue. All control tissues had low basal levels (0.3 to 2.0 pmol/h per g) of 2-5A synthetase, the enzyme responsible for 2-5A production. After mice were injected intravenously with the interferon inducer poly(I).poly(C), circulating interferon, 2-5A synthetase, and 2-5A were elevated with increasing doses of double-stranded RNA. The greatest response to poly(I).poly(C) occurred in the kidney, in which enzyme levels increased 5-fold and 2-5A levels increased 24-fold to 15 pmol/g. Mice that were infected with EMCV also possessed elevated levels of 2-5A and 2-5A synthetase in the four tissues examined, although the relative distribution differed from that observed with poly(I).poly(C), indicating that the interferon inducer affects the concentration and location of intracellular 2-5A. Brain, spleen, and kidney tissues from EMCV-infected mice contained seven- to eightfold more 2-5A than control tissues did. The nanomolar levels of 2-5A in the tissues of EMCV-infected mice provide evidence that 2-5A may play a role in the antiviral response in an intact animal. In both poly(I).poly(C)- and EMCV-treated mice, the levels of 2-5A recovered from the tissues were not directly proportional to the amount of 2-5A synthetase present. These results indicate that factors other than the level of 2-5A synthetase controlled the accumulation of 2-5A in tissues.
Cross-reactive neutralization epitopes on VP3 of human rotavirus: analysis with monoclonal antibodies and antigenic variants
Authors: Taniguchi K , Morita Y , Urasawa T , Urasawa S
Published: January 2020

We analyzed cross-reactive neutralization epitopes on protein VP3 of human rotavirus (HRV) by the use of neutralizing monoclonal antibodies (N-MAbs), which showed a variety of interserotypic reactivity patterns when examined in a neutralization test and an enzyme-linked immunosorbent assay against 15 HRV and 2 animal RV strains. Serological study with the six cross-reactive N-MAbs revealed antigenic variations in some HRV strains within the same serotype as well as a marked antigenic difference between serotype 2 strains and serotype 1, 3, and 4 strains. Epitope analysis of the antigenic variants resistant to the six individual cross-reactive N-MAbs suggested the existence of at least three distinct cross-reactive neutralization epitopes on VP3 of HRV.
A baculovirus vector can express intron-containing genes
Authors: Jeang K T , Holmgren-Konig M , Khoury G
Published: January 2020

A simjan virus 40 genomic fragment containing the genes coding for the large T and small t antigens was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the strong polyhedrin promoter. Infection of eucaryotic Spodoptera frugiperda (SF9) cells with this recombinant virus produced significant amounts of small t antigen and little or no large T protein. Analysis by Northern blotting and S1 nuclease digestion revealed correct and preferential utilization of the small t splicing signals.
Binding of the pseudorabies virus immediate-early protein to single-stranded DNA
Authors: Chlan C A , Coulter C , Feldman L T
Published: January 2020

In an attempt to correlate the ability to activate transcription with affinity for single-stranded DNA, both wild-type and temperature-sensitive pseudorabies virus immediate-early proteins were tested for the ability to bind to single-stranded DNA columns. Wild-type and temperature-sensitive immediate-early proteins bound to nonspecific single-stranded DNA columns with similar affinities at both 0 and 40 degrees C. There did not seem to be a direct correlation between the ability to activate transcription and the ability to bind to single-stranded DNA. To study further the interactions that are involved in binding to single-stranded DNA, we expressed the immediate-early protein in an Escherichia coli expression vector. In this system the expressed immediate-early protein was not phosphorylated, nor could it be complexed with mammalian cell factors. The first trp construct did not express a soluble form of the immediate-early protein, presumably due to the insoluble nature of the trp leader. We deleted a large segment of the trpE gene and found that the immediate-early fusion protein was soluble. We tested this protein for its affinity for single-stranded DNA by passage over single-stranded DNA cellulose columns. The bacterially expressed immediate-early protein bound single-stranded DNA at least as well as did the wild-type protein. Affinity for single-stranded DNA did not appear to be dependent on the phosphorylation state nor on the presence of mammalian cell factors.
Retroviral DNA integrated during infection by an integration-deficient mutant of murine leukemia virus is oligomeric
Authors: Hagino-Yamagishi K , Donehower L A , Varmus H E
Published: January 2020

Mutants with lesions engineered into the 3' region of the pol gene of murine leukemia virus (MLV), the region that encodes p40pol, have allowed us to define a function that is required for the normal integration of retroviral DNA. Two such mutants, MLV-SF1, which bears a single missense mutation (L. A. Donehower and H. E. Varmus, Proc. Natl. Acad. Sci. USA 81:6461-6465, 1984), and MLV-SF2, which has a frameshift mutation at the same site, have been used to explore the consequences of integration deficiencies. Pseudotypes that were formed with viral proteins supplied by these mutants and a replication-defective genome that carried a selectable genetic marker induced colony formation at least 100-fold less efficiently than did pseudotypes that were formed with proteins encoded by wild-type helper virus. Most of the proviruses from the mutant pseudotypes were manifestly aberrant when they were analyzed by mapping with restriction enzymes. Rare proviruses that appeared to be normal by restriction enzyme mapping were molecularly cloned and subjected to more detailed study. Two proviruses resulting from infection with the MLV-SF1 pseudotype were identical or nearly identical to wild-type MI V proviruses. Two base pairs were missing from the ends of the long terminal repeats at the host-viral junctions, and 4- or 5-base-pair duplications of host DNA flanked the proviruses. Thus, the missense mutant appears to retain a low level of normal integration activity. Four proviruses from cells that were infected with the MLV-SF2 pseudotype were flanked on both sides by viral DNA of either helper or vector origin, with a single long terminal repeat at each end of the intact vector DNA and with irregular junctions of viral and host DNAs. These results are most simply explained by the nonspecific integration of dimeric or trimeric forms of viral DNA, which may arise during infection by replicative or recombinational mechanisms.
Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene
Authors: Roebroek A J , Schalken J A , Onnekink C , Bloemers H P , Van de Ven W J
Published: January 2020

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.
Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA
Authors: Shih D S , Park I W , Evans C L , Jaynes J M , Palmenberg A C
Published: January 2020

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.
Host immune response to cytomegalovirus: products of transfected viral immediate-early genes are recognized by cloned cytolytic T lymphocytes
Authors: Koszinowski U H , Reddehase M J , Keil G M , Schickedanz J
Published: January 2020

To confirm that immediate-early (IE) genes of murine cytomegalovirus (MCMV) give rise to antigens recognized by specific cytolytic T lymphocytes (CTL), a 10.8-kilobase fragment of MCMV DNA which is abundantly transcribed at IE times was transfected into L cells expressing the Ld class I major histocompatibility glycoprotein. The viral genome fragment contains sequences of the three IE transcription units of MCMV: ie1, ie2, and ie3. In the transfected cell lines, only the predominant 2.75-kilobase transcript of ie1 and its translation product pp89 could be detected. The transfectants were analyzed for membrane expression of an IE antigen by employing clone IE1, an IE-specific CTL clone, as the probe. Only cells that expressed both the MCMV IE gene(s) and the Ld gene were recognized by the CTL clone.
Isolation of novel human retrovirus-related sequences by hybridization to synthetic oligonucleotides complementary to the tRNA(Pro) primer-binding site
Authors: Kröger B , Horak I
Published: January 2020

Synthetic oligonucleotides complementary to putative retroviral primer-binding sites were used as hybridization probes to detect novel retroviruslike sequences. An 8.1-kilobase element with structural features of a retroviral provirus was isolated from a human genomic library by this approach. Nucleotide sequence analysis of its 600-base-pair long terminal repeats revealed characteristic motifs known as regulatory signals for RNA polymerase II transcription: CCAAT, TATA, and ATTAAA. In addition, a putative pol gene displays apparent homologies to conserved regions of retroviral reverse transcriptase. The 5' long terminal repeat is flanked at its 3' end by a putative primer-binding site for reverse transcription with homology to tRNA(Pro). This element is therefore termed HuRRS-P (human retrovirus-related sequence-proline). There are 20 to 40 copies of HuRRS-P homologous sequences in DNAs of human and simian origin.