bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct helper virus requirements for Abelson murine leukemia virus-induced pre-B- and T-cell lymphomas
Authors: Poirier Y , Jolicoeur P
Published: January 2020

Abelson murine leukemia virus (A-MuLV) can induce pre-B- or T-cell lymphomas (thymomas) in mice depending on the route and time of injection. Previous studies have shown that the choice of the helper virus used to rescue A-MuLV greatly influences its ability to induce pre-B-cell lymphomas. In this study, we investigated the role of the helper virus in A-MuLV-induced thymomas. A-MuLV rescued with the helper Moloney MuLV, BALB/c endogenous N-tropic MuLV, and two chimeric MuLVs derived from these two parents were injected intrathymically in young adult NIH Swiss mice. All four A-MuLV pseudotypes were found to be equally efficient in the induction of thymomas, whereas drastic differences were observed in their pre-B-cell lymphomagenic potential. Thymoma induction by A-MuLV was independent of the replication potential of the helper virus in the thymus, and no helper proviral sequences could be detected in the majority of thymomas induced by A-MuLV rescued with parental BALB/c endogenous or chimeric MuLVs. In the thymomas in which helper proviruses were present, none of them were found integrated in the Ahi-1 region, a common proviral integration site found in A-MuLV-induced pre-B-cell lymphomas (Y. Poirer, C. Kozak, and P. Jolicoeur, J. Virol. 62:3985-3992, 1988). In addition, helper-free stocks of A-MuLV were found to be as lymphomagneic as other pseudotypes in inducing thymomas after intrathymic inoculation, in contrast to their inability to induce pre-B-cell lymphomas when injected intraperitoneally in newborn mice. Restriction enzyme analysis revealed one to three A-MuLV proviruses in each thymoma, indicating the oligoclonality of these tumors. Analysis of the immunoglobulin and T-cell receptor loci confirmed that the major population of cells of these primary thymomas belongs to the T-cell lineage. Together, these results indicate that the helper virus has no effect in the induction of A-MuLV-induced T-cell lymphomas, in contrast to its important role in the induction of A-MuLV-induced pre-B-cell lymphomas. Our data also revealed distinct biological requirements for transformation of these two target cells by v-abl.
Loss of bovine papillomavirus DNA replication control in growth-arrested transformed cells
Authors: Burnett S , Kiessling U , Pettersson U
Published: January 2020

The bovine papillomavirus type 1 (BPV-1) genome replicates as a plasmid within the nuclei of BPV-1-transformed murine C127 cells at a constant multiple copy number, and spontaneous amplification of the viral DNA is rarely observed. We report here that a mutant BPV-1 plasmid within a contact-inhibited C127 cell line replicated as a stable multicopy plasmid in exponentially growing cells but amplified to a high level in confluent cell culture. In situ hybridization analysis revealed that most of the mutant viral DNA amplification occurred in a minor subpopulation of cells within the culture. These consisted of giant nondividing cells with greatly enlarged nuclei, a cell form which was specifically induced in stationary-phase cultures. These observations indicated that expression of a viral DNA replication factor was cell growth stage specific. Consistent with this hypothesis, considerable amplification of wild-type BPV-1 DNA associated with characteristic giant cell formation was observed in typical wild-type virus-transformed C127 cultures following a period of growth arrest achieved by serum deprivation. Further observations indicated that induction of the giant-cell phenotype was dependent on BPV-1 gene expression and implicated a viral E1 replication factor in this process. Moreover, heterogeneity in virus genome copy numbers within the giant-cell population suggested a complex regulation of induction of DNA synthesis in these cells. It appears that this process represents a mechanism employed by the virus to ensure maximal viral DNA synthesis within a growth-arrested cell. Fundamental questions concerning the integration of the virus-cell control circuitry in proliferating and resting cells are discussed.
Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins
Authors: Fahnestock M L , Lewis J B
Published: January 2020

A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation.
Regulated expression of early and late RNAs and proteins from the human cytomegalovirus immediate-early gene region
Authors: Stenberg R M , Depto A S , Fortney J , Nelson J A
Published: January 2020

Expression of RNA and protein from the human cytomegalovirus immediate-early (IE) gene region (map units 0.732 to 0.751) was analyzed at early and late times after infection. The level of RNA present at late times (48 to 72 h after infection) was significantly higher than that present at IE times (5 h after infection). The profile of IE RNA in the cytoplasm of infected cells was different from that previously reported on polysomes (R. M. Stenberg, P. R. Witte, and M. F. Stinski, J. Virol. 56:665-675, 1985). The data indicate that the 1.95-kilobase (kb) major IE region 1 mRNA, which codes for the 72-kilodalton (kDa) protein, and the 1.7-kb IE region 2 (IE2) spliced mRNA, which codes for the IE2 55-kDa protein, may be preferentially associated with polysomes. However, the IE2 2.2-kb unspliced mRNA, which codes for an 86-kDa protein, may be preferentially excluded. This RNA was abundant in the cytoplasm under IE conditions but was not present on polysomes in significant quantities. This indicates that IE gene products may be involved in translational control of cytomegalovirus RNA. At late times, new transcription takes place within region 2. A 1.5-kb RNA is transcribed from a late promoter in region 2 that apparently does not function in cells infected with DNA-negative mutant ts66. These results demonstrate that the IE gene region is transcribed throughout infection and that multiple levels of regulation exist.
The cellular transcription factor E2f requires viral E1A and E4 gene products for increased DNA-binding activity and functions to stimulate adenovirus E2A gene expression
Authors: Babiss L E
Published: January 2020

Whereas a wide variety of cellular proteins interact with the cis-regulatory elements of the adenovirus E1A and E2A genes, only the DNA-binding activity of the cellular E2f factor is modulated by viral early-gene expression. An analysis of cellular E2f protein levels and adenovirus early-gene expression in a panel of independently cloned virus-transformed rodent cell lines and in virus-infected rodent cells has established that both the E1A 289-amino-acid (289R) protein and a yet-to-be-defined E4 gene product are required for maximal E2f DNA-binding activity. To distinguish between the multiple roles the E1A protein could serve in this process, the E2f DNA-binding activity was determined in a virus-transformed cell line which contains a conditional-lethal mutation affecting the 289R protein. Since E4 gene expression was not altered by the incubation conditions, the observation of reduced cellular E2f activity at the nonpermissive temperature suggests a direct role for the E1A 289R protein in E2f activation. When a virus containing a deletion in the E4 gene was introduced into cell lines which can complement the E4 gene defect, a correlation between high cellular E2f levels and increased rates of E2A gene transcription was observed. A time course analysis of the viral infection revealed that E2f functions catalytically to stimulate viral E2A gene transcription. These observations have led to several hypotheses concerning possible mechanisms by which elevated E2A gene expression, which leads to cytotoxicity, might be avoided in the transformed cell.
Bacterial lipopolysaccharide and gamma interferon induce transcription of beta interferon mRNA and interferon secretion in murine macrophages
Authors: Gessani S , Belardelli F , Pecorelli A , Puddu P , Baglioni C
Published: January 2020

Bacterial lipopolysaccharide (LPS) induces interferon (IFN) secretion and an antiviral state in murine peritoneal macrophages (PM). These cells secrete predominantly IFN-beta, as shown by neutralization assays with monoclonal antibodies. Secretion of IFN-beta is also induced in PM by IFN-gamma. LPS and IFN-gamma synergistically stimulated PM to produce IFN in amounts almost comparable to those induced by infection with Newcastle disease virus. Low levels of IFN-beta mRNA can be detected in freshly harvested PM by hybridization assays. The accumulation of this mRNA is markedly increased in PM treated with LPS or IFN-gamma, and it is further enhanced in the presence of the inhibitor of protein synthesis, cycloheximide. Similar studies were carried out on the RAW 264.7 line of transformed macrophages. These cells are induced to secrete IFN-beta by LPS but not by IFN-gamma, suggesting that this cytokine may elicit such specific response only in PM. IFN-beta mRNA is undetectable in untreated RAW 264.7 cells, and accumulation of this mRNA is induced by LPS but not by IFN-gamma. The secretion of IFN induced by these agents in PM and by LPS in RAW 264.7 cells and the corresponding accumulation of IFN-beta mRNA are blocked by an inhibitor of protein kinase C, staurosporine. The activity of this kinase is apparently necessary to stimulate accumulation of IFN-beta mRNA. The induction of IFN-beta by IFN-gamma appears to be a characteristic response of PM and may be at least in part responsible for the resistance of these cells to viral infections.
Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein
Authors: Ertl H C , Dietzschold B , Gore M , Otvos L , Larson J K , Wunner W H , Koprowski H
Published: January 2020

The T-helper cell response to the internal proteins of rabies virus was investigated. The rabies virus nucleoprotein was shown to be a major target antigen for T-helper cells that cross-react between rabies and rabies-related viruses. T-helper cells were assayed in vitro by testing virus-induced lymphocytes for lymphokine secretion in response to antigen. Immunodominant T-helper cell epitopes of the viral nucleoprotein were identified in vitro by using synthetic peptides delineated from the amino acid sequence of the nucleoprotein. The response to synthetic peptides were under Ir gene control. Antigenic peptides were tested in vivo for stimulation of rabies virus-specific T-helper cells. Inoculation of mice with peptides bearing immunodominant T-helper cell epitopes resulted in an accelerated and enhanced neutralizing antibody response upon booster immunization with inactivated rabies virus.
Chromatin structure of recombinant Moloney murine leukemia virus proviral DNAs that contain tax-responsive sequences from human T-cell lymphotropic virus type II in the presence and absence of tax
Authors: Kitado H , Fan H
Published: January 2020

Human T-cell lymphotropic virus types I and II (HTLV-I and HTLV-II) are replication-competent retroviruses which contain two additional regulatory proteins, tax and rex. tax is a transcriptional transactivator of the HTLV-I or HTLV-II long terminal repeat (LTR) and also of some heterologous promoters. To investigate the mechanism of tax transactivation, we used chimeric Moloney murine leukemia viruses (M-MuLVs) with LTRs containing tax-responsive sequences from the HTLV-II LTR (nucleotides -273 to -32). Mo+HTLV-II+ M-MuLV contained the HTLV II sequences inserted into the wild-type M-MuLV LTR at nucleotide -150, whereas delta Mo+HTLV-II+ M-MuLV contained the same sequences inserted into an M-MuLV LTR lacking its own enhancer region. HTLV-II tax (tax II)-positive mouse cells (15S-5a) infected with Mo+HTLV-II+ M-MuLV or delta Mo+HTLV-II+ M-MuLV showed higher rates of viral transcription in nuclear run-on assays than did infected tax-negative NIH 3T3 cells. The chromatin structure of these viruses was investigated by high-resolution mapping of DNase I-hypersensitive (HS) sites. Three prominent HS sites were associated with HTLV-II sequences in proviral chromatin both in tax-positive and in tax-negative cells. The spacing resembled that of the 21-base-pair (bp) repeats, but the HS sites were displaced approximately 50 bp upstream of the 21-bp repeats. This suggested that cellular proteins bound to the HTLV-II sequences in the presence or absence of tax. No direct effect of tax on chromatin structure was found. These in vivo results were consistent with results of in vitro DNase footprinting studies performed by other investigators.
Growth efficiency of naturally occurring BK virus variants in vivo and in vitro
Authors: Sugimoto C , Hara K , Taguchi F , Yogo Y
Published: January 2020

We molecularly cloned a number of BK virus (BKV) DNAs from urine samples collected from a patient with systemic lupus erythematosus undergoing immunosuppressive therapy. On the basis of the structure of the noncoding regulatory region, cloned viral DNAs were classified into a major group and several minor groups. The major group contained a single 68-base-pair (bp) promoter-enhancer element and a 63-bp sequence identified in the genomes of many BKV strains. Most of the minor groups retained a variety of duplications within the transcriptional control region and the origin of DNA replication. We assayed various cloned viral DNAs for the capacity to induce viral growth in transfected human embryonic kidney cells. While major viral DNAs induced slow viral replication, a minor viral DNA retaining partial duplication of the 68-bp element induced rapid viral growth. We concluded that reiteration of the promoter-enhancer element, which is required for efficient growth of BKV in cell culture, is not advantageous for replication of BKV in natural hosts.
Rotavirus-specific protein synthesis is not necessary for recognition of infected cells by virus-specific cytotoxic T lymphocytes
Authors: Offit P A , Greenberg H B , Dudzik K I
Published: January 2020

We found that rotavirus-specific protein synthesis was not necessary for recognition by virus-specific cytotoxic T lymphocytes (CTLs). In addition, CTLs lysed rotavirus-infected target cells prior to production of infectious virus. Target cell processing of rotavirus antigens for presentation to CTLs was enhanced by treatment of rotavirus with trypsin prior to infection; trypsin-induced cleavage of the viral hemagglutinin (vp4) has previously been found to facilitate rotavirus entry into target cells by direct penetration of virions through the plasma membrane. We conclude that sufficient quantities of exogenous viral proteins may be introduced into the cytoplasm for processing by target cells. The mechanism by which rotavirus proteins are processed for presentation to the target cell surface remains to be determined.
Neutralizing epitopes of the H5 hemagglutinin from a virulent avian influenza virus and their relationship to pathogenicity
Authors: Philpott M , Easterday B C , Hinshaw V S
Published: January 2020

To define and characterize the major neutralizing epitopes of the H5 hemagglutinin, a panel of monoclonal antibodies specific for the H5 hemagglutinin of the virulent avian influenza virus A/Turkey/Ontario/7732/66 (H5N9) was prepared. Antibodies which neutralized infectivity of the virus were used to select a panel of escape mutants. Reactivity patterns of the panel of monoclonal antibodies against the panel of mutants by both enzyme-linked immunosorbent assay serology and hemagglutination inhibition operationally defined five distinct epitopes on the H5 molecule. The mutants were analyzed in vivo for virulence in chickens, and the findings indicate that viruses with mutations in four of five epitopes were no less virulent than the wild type, producing a rapidly fatal disease, while all viruses with mutations in the fifth epitope (group 1 mutants) were attenuated. These group 1 mutants were unaltered in the cleavage properties of the hemagglutinin, suggesting that the mechanism of attenuation is unrelated to processing of the hemagglutinin. One of the group 1 mutants, 77B1v, was characterized for its ability to produce necrosis of the spleen and was found to produce none of the lesions in the spleen which are characteristic of the wild-type virus, although virus was present in this organ. The results suggest an altered tissue tropism, perhaps sparing a population of cells critical to an effective immune response.
Phorbol ester-inducible T-cell-specific expression of variant mouse mammary tumor virus long terminal repeats
Authors: Theunissen H J , Paardekooper M , Maduro L J , Michalides R J , Nusse R
Published: January 2020

Acquired proviruses of mouse mammary tumor virus (MMTV) in T-cell leukemias of male GR mice have rearrangements in the U3 region of their long terminal repeats (LTR). In contrast to the endogenous nonrearranged MMTV proviruses, these mutated copies are highly expressed in leukemic T cells. To investigate whether the sequence alterations in the LTR are responsible for the high expression of rearranged MMTV proviruses, we made constructs in which normal and variant LTRs drive the bacterial reporter gene chloramphenicol acetyltransferase (CAT). Two different rearranged LTRs were used, one containing a 420-base-pair (bp) deletion (L13) and another carrying a 456-bp deletion plus an 82-bp insertion (L42). These constructs were transfected into murine (GRSL) and human (MOLT-4) T-cell lines that either had or had not been treated with phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]). In GRSL cells, the L13-LTR-CAT construct showed transcriptional activity that was further enhanced by TPA. In MOLT-4 cells, both variant LTRs were active, but only after stimulation with TPA. In contrast, normal(N)-LTR-CAT constructs were not expressed, irrespective of TPA addition. In XC rat fibrosarcoma cells, neither normal nor variant LTRs gave rise to detectable CAT activity, either in the presence or in the absence of TPA, but dexamethasone strongly stimulated CAT activity driven by N and L42 LTRs. The L13 LTR was considerably less active, probably caused by the deletion of the distal part of the glucocorticoid responsive element. We conclude that the LTR rearrangements generate TPA responsiveness and contribute to T-cell-specific expression of MMTV variants.
Persistent human immunodeficiency virus type 1 infection of monoblastoid cells leads to accumulation of self-integrated viral DNA and to production of defective virions
Authors: Pauza C D , Galindo J
Published: January 2020

Cell-free virus preparations from persistently infected monoblastoid cells (HU937) become progressively less infectious during long-term passage. This effect is specific for cell lines derived from U937 and is not observed in persistently infected T-cell lines. Reduced infectivity is correlated with accumulation of unusual, high-molecular-weight, extrachromosomal forms of the human immunodeficiency virus type 1 (HIV-1) DNA. These DNA molecules contain multiple copies of the viral genome, and their structures are highly variable. Of 17 subclones of the HU937 cell line, 15 unique restriction fragment patterns were observed for the HIV-1 viral DNA. Structural analysis of these viral DNA species indicated that they were formed by sequential rounds of long terminal repeat-mediated integration of one circular DNA form into preexisting monomeric or multimeric structures. These viral DNA structures are termed nested self-integrates. Once formed, self-integrates prove to be stable and can be maintained for several months in culture. The unusual structures of HIV-1 DNA in persistently infected monoblastoid cells attest to an alternative to the accepted retrovirus life cycle. The self-integrated viral DNA species reported here may explain some aspects of the mechanism controlling establishment and maintenance of persistent HIV-1 infection in cells of the monocyte/macrophage lineage.
Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein
Authors: Strebel K , Klimkait T , Maldarelli F , Martin M A
Published: January 2020

We have performed a detailed analysis of the biochemical properties of the human immunodeficiency virus (HIV) type 1 vpu gene product to elucidate its function during virus replication. Our data suggest that vpu is posttranslationally modified by phosphorylation, since a 16-kilodalton phosphoprotein can be specifically immunoprecipitated with both a serum from an HIV-positive individual (HIV-positive serum) and a vpu-specific antiserum. In contrast, our results suggest that vpu is not glycosylated, even though the protein contains a potential glycosylation site. In vitro translation studies demonstrated that vpu is cotranslationally integrated into microsomal membranes, suggesting that vpu is an integral membrane protein. While vpu was found in significant quantities in virus-producing cells, the protein could not be detected in cell-free culture fluids and is therefore most likely not viron associated. Processing of viral precursor proteins was unaffected by the absence of vpu, and no differences were detected in the protein compositions of wild-type and mutant virions. However, virus release from cultures producing vpu-defective virus was found to be delayed, resulting in the intracellular accumulation of viral proteins. Our data suggest that vpu has a function in the release of virus particles from infected cells.
Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus
Authors: Corapi W V , French T W , Dubovi E J
Published: January 2020

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovine viral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulating platelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV before infection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time of infection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating platelets and a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocytopenia was severe (less than or equal to 5,000/microliters) for 1 to 6 days. In two of these three animals, extensive petechial and ecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during the period of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases of the infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. The results demonstrate that BDV infection is associated with decreases in platelet numbers and suggest that platelets may serve as carriers of circulating virus.
Examination of the roles of transcription factor Sp1-binding sites and an octamer motif in trans induction of the herpes simplex virus thymidine kinase gene
Authors: Böni J , Coen D M
Published: January 2020

Herpes simplex virus mutants with both Sp1-binding sites in the thymidine kinase (tk) promoter inactivated or an octamer motif deleted were at most modestly impaired for tk expression. Thus, no cellular transcription factor that binds upstream of the tk TATA box is solely required for trans induction of this gene.
Complete nucleotide sequence of the gene encoding VP4 of a human rotavirus (strain K8) which has unique VP4 neutralization epitopes
Authors: Taniguchi K , Nishikawa K , Urasawa T , Urasawa S , Midthun K , Kapikian A Z , Gorziglia M
Published: January 2020

In our previous study (K. Taniguchi, Y. Morita, T. Urasawa, and S. Urasawa, J. Virol. 62:2421-2426, 1987) in which the cross-reactive neutralization epitopes on VP4 of human rotaviruses were analyzed, one strain, K8, was found to bear unique VP4 neutralization epitopes. This strain, which belongs to subgroup II and serotype 1, was not neutralized by any of six anti-VP4 neutralizing monoclonal antibodies which reacted with human rotavirus strains of serotypes 1, 3, and 4 or serotypes 1 through 4. We determined the complete nucleotide sequence of the gene encoding VP4 of strain K8 by primer extension. The VP4 gene is 2,359 base pairs in length, with 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. The gene contains a long open reading frame of 2,325 bases capable of coding for a protein of 775 amino acids. When compared with those of other human rotaviruses, VP4 of strain K8 had an insertion of one amino acid after residue 135, as found in simian rotavirus strains, and in addition, it had a deletion of one amino acid (residue 575). The amino acid homology of VP4 of strain K8 and those of other virulent human rotaviruses was only 60 to 70%. This was unusual, since over 90% VP4 homology has been found among the other virulent human rotavirus strains. In contrast, the VP7 amino acid sequence of the K8 strain was quite similar (over 98% homology) to those of other serotype 1 human rotaviruses. Thus, the K8 strain appears to have a unique VP4 gene previously not described.
Mutations in VP1 of poliovirus specifically affect both encapsidation and release of viral RNA
Authors: Kirkegaard K
Published: January 2020

The phenotypic defects of two type 1 Mahoney poliovirus mutants, termed VP1-101 and VP1-102, were caused by two different small deletions in the region of the RNA genome encoding the amino terminus of the capsid protein VP1. This portion of VP1 was unresolved in the three-dimensional structure of the poliovirion, buried within the virion, and likely to interact with the viral RNA. Both VP1-101 and VP1-102 showed a diminished ability to enter CV1 but not HeLa cells; both mutants formed plaques on CV1 and HeLa cells that were smaller than wild type. Neither the rate of binding to cells nor the rate of subsequent receptor-dependent conformational change of the mutant poliovirions was affected. However, both mutants displayed delayed kinetics of RNA release compared with wild-type virus. One of the mutants, VP1-102, also displayed a defect in viral morphogenesis: 75S empty capsids formed normally, but 150S particles that contained RNA accumulated much more slowly. We suggest that the VP1-102 mutation affects RNA encapsidation as well as RNA release, whereas the VP1-101 mutation affects only RNA release. Therefore, RNA packaging and RNA release are genetically linked but can be mutated separately in different VP1 alleles, and both processes involve the amino terminus of VP1.
Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions
Authors: Ghisotti D , Finkel S , Halling C , Dehò G , Sironi G , Calendar R
Published: January 2020

We sequenced the leftmost 2,640 base pairs of bacteriophage P4 DNA, thus completing the sequence of the 11,627-base-pair P4 genome. The newly sequenced region encodes three nonessential genes, which are called gop, beta, and cII (in order, from left to right). The gop gene product kills Escherichia coli when the beta protein is absent; the gop and beta genes are transcribed rightward from the same promoter. The cII gene is transcribed leftward to a rho-independent terminator. Mutation of this terminator creates a temperature-sensitive phenotype, presumably owing to a defect in expression of the beta gene.
Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey
Authors: Shibata R , Miura T , Hayami M , Sakai H , Ogawa K , Kiyomasu T , Ishimoto A , Adachi A
Published: January 2020

We constructed a full-length molecular clone of simian immunodeficiency virus from an African green monkey. Upon transfection, this clone directed the production of virus particles cytopathic and infectious to human CD4+ leukemia cell lines. Mutations were introduced by recombinant DNA techniques into eight open reading frames of simian immunodeficiency virus from the African green monkey thus far identified. The phenotypes of mutant viruses, i.e., infectivity, cytopathogenicity, transactivation of gene expression controlled by a long terminal repeat, and viral RNA and protein syntheses, were examined by transfection and infection experiments. Three structural (gag, pol, and env) and two regulatory (tat and rev) gene mutants were not infectious, whereas vif, vpx, and nef were dispensable for infectivity and mutant viruses were highly cytopathic. In transient transfection assays, a rev mutant produced mainly small mRNA species and no detectable virus protein and particles. The transactivation potential of a tat mutant was about 10-fold less than that of wild-type DNA, generating small amounts of virus.
A subpopulation of the avian erythroblastosis virus v-erbA protein, a member of the nuclear hormone receptor family, is glycosylated
Authors: Privalsky M L
Published: January 2020

The v-erbA oncogene of avian erythroblastosis virus is derived from a cellular gene for a thyroid hormone (T4/T3 thyronine) receptor and encodes a DNA-binding protein found principally in the nucleus of the infected cell. I report here that a subpopulation of the v-erbA protein is glycosylated. The v-erbA protein, therefore, is another member of the newly recognized family of eucaryotic transcription factors and related polypeptides which are glycoproteins.
Human cytomegalovirus induces expression of cellular topoisomerase II
Authors: Benson J D , Huang E S
Published: January 2020

Previous work from our laboratory has suggested that topoisomerase II is required for replication of human cytomegalovirus (HCMV). In assays of confluent human embryonic lung cells infected with HCMV, topoisomerase II inhibitors exhibited an irreversible inhibition of viral DNA replication. However, Northern (RNA blot) and Western (immunoblot) analyses of confluent uninfected human embryonic lung cells detected very low levels of cellular topoisomerase II RNA and protein. Quantitation of human topoisomerase II RNA and protein levels at various times after HCMV infection revealed that HCMV induces increased intracellular levels of both topoisomerase II RNA and protein. Such accumulation began at early times of infection, continued through late in infection, and was not reduced by inhibition of viral DNA synthesis. This is the first report of such induction by a viral infection. Topoisomerase II was also detected in isolated HCMV virions.
Oligomers of the cytoplasmic domain of the p62/E2 membrane protein of Semliki Forest virus bind to the nucleocapsid in vitro
Authors: Metsikkö K , Garoff H
Published: January 2020

We analyzed the interaction between the nucleocapsid and synthetic peptides corresponding to the complete or truncated cytoplasmic protein domain of the Semliki Forest virus p62/E2 glycoprotein. We found that the peptide corresponding to the full-length domain efficiently bound nucleocapsids when coupled to a solid matrix via specific antibodies, whereas the shorter one did not. In solution, a substantial fraction of the full-length peptide associated into oligomers. Binding studies showed that it was mostly these oligomers, rather than the monomeric form of the peptide, which were able to interact with the nucleocapsid. Thus, our findings demonstrate a direct interaction between the spike proteins and the viral nucleocapsid. Furthermore, they suggest that this interaction is directed through formation of complexes containing several p62 or E2 subunits.
A major portion of the latent pseudorabies virus genome is transcribed in trigeminal ganglia of pigs
Authors: Priola S A , Gustafson D P , Wagner E K , Stevens J G
Published: January 2020

Pseudorabies virus (PRV) is a porcine herpesvirus that establishes latent infections in trigeminal ganglia. To determine whether PRV expresses any transcripts that could play a role in latency, the trigeminal ganglia of 14 pigs previously inoculated through the nose and latently infected with PRV(Ka) were assayed by in situ nucleic acid hybridization for the presence of PRV-specific RNA. Hybridizations employing probes encompassing the entire viral genome revealed that an area extending from 0.64 to 0.82 map units was transcriptionally active. The DNA probe that most consistently detected transcripts was BamHI-8, a fragment which contains the gene for the immediate-early protein. With this probe, ganglia from 10 (71%) of 14 pigs scored positive for PRV RNA, although only 1 (8%) of 12 of the ganglia from the opposite side reactivated virus after explanation and culture of latently infected trigeminal ganglia. The RNA was transcribed from the strand opposite to that coding for the immediate-early protein; the signal was neuronally localized, with dense nuclear accumulation accompanied by variable numbers of grains over the cytoplasm. Northern RNA blot analysis showed that a discrete set of poly(A)- PRV transcripts were present in latently infected trigeminal ganglia. Additional in situ nucleic acid hybridization analysis revealed that the 3' limit of the transcriptionally active area was located in a 1.2-kilobase fragment upstream and adjacent to the 5' end of the immediate-early protein RNA, whereas the 5' limit was as much as 4.9 kilobases downstream from the 3' end of this RNA. PRV therefore expresses latent-phase transcripts that, although similar in many respects to latent-phase transcripts reported for other herpesviruses, have some unique properties.
Transforming growth factors beta 1 and 2 transcriptionally regulate human papillomavirus (HPV) type 16 early gene expression in HPV-immortalized human genital epithelial cells
Authors: Woodworth C D , Notario V , DiPaolo J A
Published: January 2020

Human papillomavirus type 16 (HPV16) early proteins E6 and E7 have been implicated in maintenance of the malignant phenotype in cervical cancer. Transforming growth factors beta one and two (TGF betas 1 and 2), polypeptides that regulate cellular growth and differentiation, reversibly inhibited expression of the HPV16 E6 and E7 genes in several immortal genital epithelial cell lines. Loss of E6 and E7 protein expression followed a dramatic time- and dose-dependent decrease in E6 and E7 RNA levels and was accompanied by cessation of cell proliferation. TGF betas 1 and 2 inhibited HPV16 RNA expression at the transcriptional level; inhibition was dependent upon ongoing protein synthesis. TGF betas 1 and 2 also induced a six- to sevenfold increase in TGF beta 1 RNA. Cells became partially resistant to the inhibitory effects of TGF beta 1 on cell growth and HPV early gene expression after prolonged cultivation in vitro or after malignant transformation. Thus, TGF beta 1 may function as an autocrine regulator of HPV gene expression in infected genital epithelial cells.
Murine coronavirus nonstructural protein ns2 is not essential for virus replication in transformed cells
Authors: Schwarz B , Routledge E , Siddell S G
Published: January 2020

Two isolates of the murine hepatitis virus (MHV) strain JHM, which differed in their ability to express the nonstructural gene product ns2, were characterized. The MHV Wb3 isolate encodes a 30,000-molecular-weight ns2 protein that can be readily detected in infected cells by using a specific monoclonal antibody, MAb 2A. The MHV Wb1 isolate is a deletion mutant that lacks a functional ns2 gene and the transcriptional signals required for the synthesis of an ns2 mRNA. However, there are no obviously significant differences in the growth of the MHV Wb1 and MHV Wb3 isolates in continuous cell lines or in the synthesis of viral mRNAs or proteins in infected cells. These results demonstrate that the ns2 gene product is not essential for MHV replication in transformed murine cells and suggests that the function of the ns2 gene may only be manifest in vivo.
Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector
Authors: Höss A , Moarefi I , Scheidtmann K H , Cisek L J , Corden J L , Dornreiter I , Arthur A K , Fanning E
Published: January 2020

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.
Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer
Authors: Kamachi Y , Ogawa E , Asano M , Ishida S , Murakami Y , Satake M , Ito Y , Shigesada K
Published: January 2020

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.
Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus
Authors: Gorman O T , Donis R O , Kawaoka Y , Webster R G
Published: January 2020

Phylogenetic analysis of 20 influenza A virus PB2 genes showed that PB2 genes have evolved into the following four major lineages: (i) equine/Prague/56 (EQPR56); (ii and iii) two distinct avian PB2 lineages, one containing FPV/34 and H13 gull virus strains and the other containing North American avian and recent equine strains; and (iv) human virus strains joined with classic swine virus strains (i.e., H1N1 swine virus strains related to swine/Iowa/15/30). The human virus lineage showed the greatest divergence from its root relative to other lineages. The estimated nucleotide evolutionary rate for the human PB2 lineage was 1.82 x 10(-3) changes per nucleotide per year, which is within the range of published estimates for NP and NS genes of human influenza A viruses. At the amino acid level, PB2s of human viruses have accumulated 34 amino acid changes over the past 55 years. In contrast, the avian PB2 lineages showed much less evolution, e.g., recent avian PB2s showed as few as three amino acid changes relative to the avian root. The completion of evolutionary analyses of the PB1, PB2, PA and NP genes of the ribonucleoprotein (RNP) complex permits comparison of evolutionary pathways. Different patterns of evolution among the RNP genes indicate that the genes of the complex are not coevolving as a unit. Evolution of the PB1 and PB2 genes is less correlated with host-specific factors, and their proteins appear to be evolving more slowly than NP and PA. This suggests that protein functional constraints are limiting the evolutionary divergence of PB1 and PB2 genes. The parallel host-specific evolutionary pathways of the NP and PA genes suggest that these proteins are coevolving in response to host-specific factors. PB2s of human influenza A viruses share a common ancestor with classic swine virus PB2s, and the pattern of evolution suggests that the ancestor was an avian virus PB2. This same pattern of evolution appears in the other genes of the RNP complex. Antigenic studies of HA and NA proteins and sequence comparisons of NS and M genes also suggest a close ancestry for these genes in human and classic swine viruses. From our review of the evolutionary patterns of influenza A virus genes, we propose the following hypothesis: the common ancestor to current strains of human and classic swine influenza viruses predated the 1918 human pandemic virus and was recently derived from the avian host reservoir.
Mutational analysis of the resolution sequence of vaccinia virus DNA: essential sequence consists of two separate AT-rich regions highly conserved among poxviruses
Authors: Merchlinsky M
Published: January 2020

In replicative forms of vaccinia virus DNA, the unit genomes are connected by palindromic junction fragments that are resolved into mature viral genomes with hairpin termini. Bacterial plasmids containing the junction fragment for vaccinia virus or Shope fibroma virus were converted into linear minichromosomes of vector sequence flanked by poxvirus hairpin loops after transfection into infected cells. Analysis of a series of symmetrical deletion mutations demonstrated that in vaccinia virus the presence of the DNA sequence ATTTAGTGTCTAGAAAAAAA on both sides of the apical segment of the concatemer junction is crucial for resolution. To determine the precise architecture of the resolution site, a series of site-directed mutations within this tract of nucleotides were made and the relative contribution of each nucleotide to the efficaciousness of resolution was determined. The nucleotide sequence necessary for the resolution of the vaccinia virus concatemer junction, (A/T)TTT(A/G)N7-9AAAAAAA, is highly conserved among poxviruses and found proximal to the hairpin loop in the genomes of members of the Leporipoxvirus, Avipoxvirus, and Capripoxvirus genera.
Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells
Authors: Vink C , van Gent D C , Plasterk R H
Published: January 2020

An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.
Complement-mediated, infection-enhancing antibodies in plasma from vaccinated macaques before and after inoculation with live simian immunodeficiency virus
Authors: Montefiori D C , Murphey-Corb M , Desrosiers R C , Daniel M D
Published: January 2020

Rhesus monkeys vaccinated against infection with simian immunodeficiency virus (SIV) were examined, in retrospect, for the presence of infection-enhancing antibodies and possible consequences associated with the presence of these antibodies. At the time of experimental inoculation with live virus, complement-mediated, infection-enhancing antibodies were detected in plasma specimens from six of six animals vaccinated with detergent-inactivated whole virus, from nine of nine animals vaccinated with Formalin-inactivated whole virus, and from seven of eight animals immunized with two SIV subunit preparations. The presence of infection-enhancing antibodies at the time of viral challenge gave no indication of predicting vaccine success or failure. After live-virus challenge, titers of infection-enhancing antibodies, like enzyme-linked immunosorbent assay titers, increased in unprotected animals and decreased or became undetectable in animals protected by vaccination. Thus, vaccine protection against SIV infection can still be achieved in the presence of detectable complement-mediated, infection-enhancing antibodies.
Identification of cis-acting repressive sequences within the negative regulatory element of human immunodeficiency virus type 1
Authors: Lu Y C , Touzjian N , Stenzel M , Dorfman T , Sodroski J G , Haseltine W A
Published: January 2020

The negative regulatory element of human immunodeficiency virus type 1 is a 260-nucleotide-long sequence that decreases the rate of RNA transcription initiation specified by the long terminal repeat. This region has the potential to bind several cellular transcription factors. Here it is shown that sequences which recognize the NFAT-1 and USF cellular transcription factors contribute to this negative regulatory effect. The sequences within the negative regulatory element which resemble the AP-1 site and the URS do not negatively regulate human immunodeficiency virus long terminal repeat transcription initiation.