bluuebunny/crossref_metadata_2025 huggingface dataset:
A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior
Authors: Freund R , Garcea R L , Sahli R , Benjamin T L
Published: January 2020

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.
Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein
Authors: Dillon P J , Nelbock P , Perkins A , Rosen C A
Published: January 2020

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.
Generation of recombinant murine retroviral genomes containing the v-src oncogene: isolation of a virus inducing hemangiosarcomas in the brain
Authors: Hevezi P , Goff S P
Published: January 2020

A series of recombinant retroviral genomes was generated by cotransformation of NIH 3T3 cells with a mixture of cloned DNAs: a proviral copy of the wild-type Moloney murine leukemia virus, and Moloney-based vectors containing defective copies of the chicken v-src and the murine v-abl oncogenes. Morphologically transformed foci, appearing at low frequencies in these cultures, released high titers of transforming viruses. Analysis of one group of these viruses showed that the genomes were recombinants containing portions of the viral gag gene juxtaposed to the v-src oncogene. Biologically active cloned DNAs of two of these viruses were obtained and mapped in detail. One of these viruses did not cause disease after inoculation into newborn mice, but the other induced rapidly fatal hemangiosarcomas located exclusively in the brain.
Polymerase chain reaction analysis of defective human T-cell leukemia virus type I proviral genomes in leukemic cells of patients with adult T-cell leukemia
Authors: Korber B , Okayama A , Donnelly R , Tachibana N , Essex M
Published: January 2020

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia, and the clonally derived leukemic cells all contain proviral genomes. Polymerase chain reaction with a variety of primers which span the HTLV-I genome was used to determine that a significant fraction of patients (at least 32%) carry deleted viral genomes in their leukemic cells. The pX region of the HTLV-I genome encoding the regulatory genes tax and rex was preferentially retained. The fact that the tax coding region was retained provides supporting evidence that the tax protein contributes to leukemogenesis in vivo. The reasonably high fraction of patients with adult T-cell leukemia carrying deleted genomes in their tumor cells suggests that the deletions have a role in leukemogenesis.
Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins
Authors: Ito T , Gorman O T , Kawaoka Y , Bean W J , Webster R G
Published: January 2020

Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180
Authors: Reuter S , Delius H , Kahn T , Hofmann B , zur Hausen H , Schwarz E
Published: January 2020

The human cervical carcinoma cell line ME180 was examined for human papillomavirus (HPV) DNA and RNA. The integrated DNA of a presumably new HPV type showing a relationship closer to HPV39 than to HPV18 was cloned and sequenced. HPV sequences from the E6-E7-E1 region are expressed as poly(A)+ RNAs.
Identification of the human herpesvirus 6 glycoprotein H and putative large tegument protein genes
Authors: Josephs S F , Ablashi D V , Salahuddin S Z , Jagodzinski L L , Wong-Staal F , Gallo R C
Published: January 2020

Determination of the nucleotide sequences of two molecular clones of human herpesvirus 6 (HHV-6) (strain GS) and comparison with those of human cytomegalovirus (HCMV) has allowed the identification of the genes for the glycoprotein H (gH) and the putative large tegument protein of HHV-6. Two molecular clones of fragments of HHV-6, the BamHI-G fragment (7,981 bp) of the clone termed pZVB43 and a HindIII fragment (8,717 bp) of the clone termed pZVH14, represent approximately 10% of the HHV-6 genome (16,689). An open reading frame within the BamHI-G fragment was designated the gH gene of HHV-6 because of the extensive sequence similarity of its predicted product (79,549 Da) to the HCMV gH gene product. The predicted product (239,589 Da) of an open reading frame within clone pZVH14 showed homology to the predicted product of the proposed gene of HCMV representing the large tegument protein. Computer analyses indicated a closer relationship of the predicted peptides of these HHV-6 genes to those of HCMV than to those of the other human herpesviruses Epstein-Barr virus, herpes simplex virus type 1, and varicella-zoster virus. The gH gene was more conserved among the human herpesvirus group, while significant sequence similarity of the tegument gene could be found only with that of HCMV. The data reported here with one conserved gene (gH) and a more divergent gene (tegument) support previous reports that HHV-6 and HCMV are more closely related to each other than to the other well-characterized human herpesviruses.
Isolation of a type D retrovirus from B-cell lymphomas of a patient with AIDS
Authors: Bohannon R C , Donehower L A , Ford R J
Published: January 2020

An atypical syncytial variant of a high-grade Burkitt's-type B-cell lymphoma from a patient with AIDS who was seropositive for human immunodeficiency virus type 1 was studied. A productive type D retrovirus infection was identified in early-passage cell lines derived from two lymphomas from this patient. Nucleotide and amino acid sequence analysis as well as immunologic reactivity indicated that the isolated virus was highly related to Mason-Pfizer monkey virus (MPMV). MPMV is an immunosuppressive type D retrovirus that causes an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by polymerase chain reaction generated the appropriate MPMV-specific fragments and indicated that the patient was infected with the MPMV-like retrovirus. In addition, the patient's serum contained antibodies which recognized type D viral env proteins (gp70 and gp20) and gag proteins (p27 and p14). Although there have been reports of human cell lines infected with type D retroviruses and of type D-reactive human sera, this is the first evidence of a type D retrovirus infection in a human confirmed by virus isolation, serum reactivity, and viral DNA identification in tumor tissue.
Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential
Authors: Jones T R , Muzithras V P , Gluzman Y
Published: January 2020

The US6 gene family, located within the unique short region (US) of the human cytomegalovirus (HCMV) genome, contains six open reading frames (US6 through US11) which may encode glycoproteins, such as gcII (D. Gretch, B. Kari, R. Gehrz, and M. Stinski, J. Virol. 62:1956-1962, 1988). By homologous recombination, several different recombinant HCMV were created which contain a marker gene, beta-glucuronidase, inserted within this gene family. It was demonstrated that beta-glucuronidase has utility as a marker gene for the identification of recombinants in this herpesvirus system, without the occurrence of deletions in other regions of the viral genome. DNA and RNA blot analyses attested to the fidelity of the recombination. Immunoprecipitation experiments using monospecific polyclonal antisera indicated that the US10 and/or US11 gene products were not expressed in the recombinants, as predicted. These results, along with single-cycle growth analyses, indicated that the US10 and US11 gene products are nonessential for virus replication and growth in tissue culture. HCMV recombinants expressing beta-glucuronidase seemed to be genetically stable.
Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies
Authors: Nicholson R , Pelletier J , Le S Y , Sonenberg N
Published: January 2020

The naturally uncapped genomic and mRNAs of poliovirus initiate translation by an internal ribosome-binding mechanism. The mRNA 5' untranslated region (UTR) of poliovirus is approximately 750 nucleotides in length and has seven to eight (depending on the serotype) AUG codons upstream of the initiator AUG. The sequence required for internal ribosome binding has been termed the ribosome landing pad (RLP). To better understand the mechanisms of internal initiation, we have determined the boundaries and critical elements of the RLP of poliovirus type 2 (Lansing strain) in vivo. By using deletion analysis, we demonstrate the existence of a core RLP in the poliovirus mRNA 5' UTR whose boundaries are between nucleotides 134 and 155 at the 5' end and nucleotides 556 and 585 at the 3' end. Sequences flanking the core RLP affect translational activity. The importance of several stem-loop structures in the RLP for internal initiation has been determined. Mutation of the phylogenetically conserved loop sequences in the proximal stem-loop structure of the RLP (stem-loop structure III; nucleotides 127 to 165) abolished internal translation. However, deletion of the second stem-loop in the RLP (stem-loop structure IV; nucleotides 189 to 223) reduced internal translation by only 50%. Internal deletions encompassing nucleotides 240 to 300, 350 to 380, or 450 to 480, predicted to disrupt stem-loop structure V and possibly VI, also abrogated internal initiation. Small point mutations within a short polypyrimidine sequence, highly conserved among all picornaviruses, abolished translation. A conservation of distance between the conserved polypyrimidine tract and a downstream AUG could play an important role in the mechanism of internal initiation.
Helix-loop-helix transcriptional activators bind to a sequence in glucocorticoid response elements of retrovirus enhancers
Authors: Corneliussen B , Thornell A , Hallberg B , Grundström T
Published: January 2020

A family of nuclear proteins, designated SL3-3 enhancer factors 2 (SEF2), were found to interact with an Ephrussi box-like motif within the glucocorticoid response element in the enhancer of the murine leukemia virus SL3-3. Mutation of the DNA sequence decreased the basal enhancer activity in various cell lines. The important nucleotides for binding of SEF2 are conserved in most type C retroviruses. Various cell types displayed differences both in the sets of SEF2-DNA complexes formed and in their amounts. A cDNA which encoded a protein that interacted specifically with the SEF2-binding sequence was isolated from human thymocytes. The nucleotide sequence specificity of the recombinant protein, expressed in Escherichia coli, corresponded to that of at least one of the nuclear SEF2 proteins. Sequence analysis of the cDNA revealed that it belongs to the basic helix-loop-helix class of DNA-binding proteins. Several mRNA transcripts of different sizes were identified. Molecular analysis of cDNA clones revealed multiple related mRNA species containing alternative coding regions, which are most probably a result of differential splicing.
Human cytomegalovirus productively infects primary differentiated macrophages
Authors: Ibanez C E , Schrier R , Ghazal P , Wiley C , Nelson J A
Published: January 2020

Monocytes are one of the predominant cell types in the peripheral blood that are infected by human cytomegalovirus (HCMV). Although virus can be detected in these cells in vivo, HCMV replication in cultured monocytes has been unsuccessful. In this study, we demonstrate efficient HCMV replication in cultured monocytes. HCMV permissiveness in these cells was dependent on nonadherent cell-induced stimulation of the monocyte, with subsequent morphological differentiation into macrophages. Approximately 40% of the cells infected by virus were detected by immunofluorescent staining with both immediate-early and late antibodies. In addition, viral plaque assays demonstrated significant productive infection of macrophages. These observations are consistent with the suggestion that the monocyte/macrophage serves as a source of viral amplification and dissemination.
Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells
Authors: Band V , De Caprio J A , Delmolino L , Kulesa V , Sager R
Published: January 2020

We have shown previously that introduction of the human papillomavirus type 16 (HPV16) or HPV18 genome into human mammary epithelial cells induces their immortalization. These immortalized cells have reduced growth factor requirements. We report here that transfection with a single HPV16 gene E6 is sufficient to immortalize these cells and reduce their growth factor requirements. The RB protein is normal in these cells, but the p53 protein is sharply reduced, as shown by immunoprecipitation with anti-p53 antibody (pAB 421). We infer that the E6 protein reduces the p53 protein perhaps by signalling its destruction by the ubiquitin system. The HPV-transforming gene E7 was unable to immortalize human mammary epithelial cells. Thus, cell-specific factors may determine which viral oncogene plays a major role in oncogenesis.
Characterization of the hepatitis B virus EnhI enhancer and X promoter complex
Authors: Guo W T , Bell K D , Ou J H
Published: January 2020

The hepatitis B virus EnhI enhancer element overlaps the promoter of the X gene. By performing methylation interference experiments, four protein factor binding sites clustered in a 120-bp region were found to control the EnhI enhancer and X promoter activities. Deletion mapping experiments indicated that the two upstream protein factor binding sites constituted a basal enhancer module. This module, likely bound by a liver-specific factor and a ubiquitous factor, could activate the herpes simplex virus thymidine kinase gene promoter by 5- or 10-fold, depending on the orientation, in Huh7 cells, a liver-derived cell line, but not in other cell types tested. The two downstream protein factor binding sites interact with the upstream basal enhancer module in an orientation- and distance-dependent manner to increase the enhancer activity by another 10-fold. In addition, at least one of the two downstream protein factor binding sites is also essential for the X promoter activity.
Evidence for a chromosomal location of polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus
Authors: Xu D M , Stoltz D
Published: January 2020

Evidence is presented in support of a chromosomal location for sequences homologous to polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus. In this study, four different viral genome segments were cloned and used as probes against genomic DNA extracted from male parasitoids and digested with a variety of restriction enzymes. Each probe typically identified a single off-size fragment (OSF) in the case of enzymes not cutting viral genome segments, while two OSFs were generated by enzymes cutting at one and two sites. While extra OSFs were occasionally observed, these were invariably found to be due to the presence of polymorphic restriction sites in flanking chromosomal DNA. Analysis of these data suggests that a single, stable chromosomal locus exists for sequences homologous to each viral genome segment; the data also indicate that viral and cognate parasitoid genomic DNAs are largely if not entirely colinear.
Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant
Authors: Li G P , Prágai B M , Rice C M
Published: January 2020

A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro. Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus. In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized. Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized. Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells. However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed. Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3. These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus.
Immunological elimination of infected cells as the candidate mechanism for tumor protection in polyomavirus-infected mice
Authors: Wirth J J , Fluck M M
Published: January 2020

The uniformly lethal development of mammary tumors in polyomavirus-infected adult female nude mice was prevented by adoptive cell transfer of polyomavirus-immune splenocytes or peritoneal cells. Transferred immune cells also lowered the growth rate of emerging tumors. The induction of other relatively less frequent tumors of the skin and bone was decreased as well. Using in situ hybridization of whole-body sections as well as hybridization of nucleic acids from the mammary glands, we show for the first time that transferred immune cells, but not normal cells, virtually eliminated virus signal in the whole mouse and in the mammary glands. Since infected and tumorous mammary glands produce very little infectious virus, it appears that a major mechanism mediating the prevention of polyomavirus oncogenesis involves the immunological elimination of nonproductively and persistently infected cells.
Variation in simian immunodeficiency virus env is confined to V1 and V4 during progression to simian AIDS
Authors: Overbaugh J , Rudensey L M , Papenhausen M D , Benveniste R E , Morton W R
Published: January 2020

We have monitored changes in the simian immunodeficiency virus (SIV) envelope (env) gene in two macaques which developed AIDS after inoculation with a molecular clone of SIV. As the animals progressed to AIDS, selection occurred for viruses with variation in two discrete regions (V1 and V4) but not for viruses with changes in the region of SIV env that corresponds to the immunodominant, V3 loop of human immunodeficiency virus. Within the highly variable domains, the vast majority of nucleotide changes encoded an amino acid change (98%), suggesting that these envelope variants had evolved as a result of phenotypic selection. Analysis of the biological properties of these variants, which have been selected for in the host, may be useful in defining the mechanisms underlying viral persistence and progression to simian AIDS.
Analysis of equine humoral immune responses to the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus
Authors: Chong Y H , Ball J M , Issel C J , Montelaro R C , Rushlow K E
Published: January 2020

Defined segments of the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus were expressed as TrpLE fusion proteins and examined for their reactivity in Western immunoblots against a diverse panel of equine immune sera. The most immunogenic region of gp45 was localized to its amino terminus, positioned between the hydrophobic fusion and the transmembrane domains. A series of overlapping synthetic peptides were used in enzyme-linked immunosorbent assays to define an immunodominant epitope within this region. In contrast, the carboxy-terminal half of gp45 displayed both weak and variable immunoreactivity with equine immune sera.
A second-site revertant of a defective herpes simplex virus ICP4 protein with restored regulatory activities and impaired DNA-binding properties
Authors: Shepard A A , DeLuca N A
Published: January 2020

A mutant of herpes simplex virus type 1, vi12, encodes a DNA-binding- and transactivation-deficient ICP4 polypeptide. Because of the mutation, the vi12 virus does not grow on Vero cells but must be propagated on cells that express complementing levels of wild-type ICP4 (E5 cells). A pseudorevertant of vi12, designated pri12, was isolated on the basis of the restored ability to replicate on Vero cells. In addition to the original i12 insertion mutation at amino acid 320, the ICP4 molecule expressed from pri12 possesses an alanine to valine substitution at amino acid 342 within the ICP4 gene. The infectivity of pri12 on Vero cells as measured by burst size is elevated by 5 orders of magnitude relative to that observed for vi12, reflecting the restored ability of the mutant ICP4 molecule possessing the alanine to valine substitution to activate transcription and thus support viral replication. Despite the restored regulatory activities of the pri12 ICP4 molecule, the ability of the pseudorevertant ICP4 molecule to form a high-affinity, specific interaction with the consensus binding site was still impaired relative to that of wild-type ICP4. This observation suggests that the in vitro-measured DNA-binding properties of ICP4 may not reflect the functional interactions occurring in vivo that mediate transcriptional activation.
Chloroquine enhances replication of Semliki Forest virus and encephalomyocarditis virus in mice
Authors: Maheshwari R K , Srikantan V , Bhartiya D
Published: January 2020

Chloroquine (CHL) has been suggested to play an important role in the development of Burkitt's lymphoma by enhancing Epstein-Barr virus expression. Herpes zoster virus incidence is markedly increased following malaria infection in children being treated with CHL. Recently, CHL has also been shown to dramatically increase the transactivation of Tat protein purified from human immunodeficiency virus. These previous studies indirectly suggest that CHL may be involved in the enhancement of virus replication. This study demonstrates for the first time that CHL indeed enhances Semliki Forest virus and encephalomyocarditis virus replication in mice. These results raise the possible connection between the increased spread of AIDS in endemic malaria areas and the wide use of CHL in those areas for the chemotherapy of malaria.
Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity
Authors: Herold B C , WuDunn D , Soltys N , Spear P G
Published: January 2020

The purpose of this study was to identify the herpes simplex virus glycoprotein(s) that mediates the adsorption of virions to cells. Because heparan sulfate moieties of cell surface proteoglycans serve as the receptors for herpes simplex virus adsorption, we tested whether any of the viral glycoproteins could bind to heparin-Sepharose in affinity chromatography experiments. Two glycoproteins, gB and gC, bound to heparin-Sepharose and could be eluted with soluble heparin. In order to determine whether virions devoid of gC or gB were impaired for adsorption, we quantitated the binding of wild-type and mutant virions to cells. We found that at equivalent input concentrations of purified virions, significantly fewer gC-negative virions bound to cells than did wild-type or gB-negative virions. In addition, the gC-negative virions that bound to cells showed a significant delay in penetration compared with wild-type virus. The impairments in adsorption and penetration of the gC-negative virions can account for their reduced PFU/particle ratios, which were found to be about 5 to 10% that of wild-type virions, depending on the host cell. Although gC is dispensable for replication of herpes simplex virus in cell culture, it clearly facilitates virion adsorption and enhances infectivity by about a factor of 10.
Characterization of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18
Authors: Jenison S A , Yu X P , Valentine J M , Galloway D A
Published: January 2020

We have previously reported that the most common human serum immunoglobulin G antibody reactivities to human papillomavirus type 16 and type 18 (HPV16 and HPV18)-encoded proteins are directed against the minor capsid proteins (HPV16 L2 and HPV18 L2) and to the E7 protein of HPV16 (S. A. Jenison, X.-P. Yu, J. M. Valentine, L. A. Koutsky, A. E. Christiansen, A. M. Beckmann, and D. A. Galloway, J. Infect. Dis. 162:60-69, 1990). In this study, the antibody-reactive segments of the HPV16 E7, HPV16 L2, and HPV18 L2 polypeptides were mapped by using nested sets of deleted recombinant proteins. A single major immunoreactive region was identified in the HPV16 E7 polypeptide between amino acids (aa) 21 and 34 (DLYCYE-QLNDSSEE). In contrast, three distinct immunoreactive regions of the HPV16 L2 polypeptide were present in the segment between aa149 and aa204, and three distinct immunoreactive regions of the HPV18 L2 polypeptide were present in the segment between aa110 and aa211. With the exception of one serum sample, serum immunoglobulin G antibodies which reacted with HPV16 L2 polypeptides or with HPV18 L2 polypeptides were not cross-reactive.
Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA
Authors: Staprans S , Loeb D D , Ganem D
Published: January 2020

Hepadnaviruses replicate their circular DNA genomes via reverse transcription of an RNA intermediate. The initial product of reverse transcription, minus-strand DNA, contains two copies of a short direct repeat (DR) sequence, termed DR1 and DR2. Plus-strand DNA synthesis initiates at DR2 on minus-strand DNA, using as a primer a short, DR1-containing oligoribonucleotide derived by cleavage and translocation from the 5' end of pregenomic RNA. To clarify the sequence requirements for plus-strand primer cleavage and translocation, we have constructed mutants of the duck hepatitis B virus bearing base changes in or around the DR1 sequence in the primer. A point mutation at the terminal nucleotide of DR1 has a striking phenotype: normal levels of duplex viral DNA are produced, but nearly all of the DNA is linear rather than circular. Mapping of the 5' end of plus-strand DNA reveals that primer cleavage occurs with normal efficiency and accuracy, but the primer is not translocated to DR2; rather, it is extended in situ to generate duplex linear DNA. Other mutations just 3' to DR1 similarly affect primer translocation, although with differing efficiencies. Linear DNA found in wild-type virus preparations has the same fine structure as the mutant linears described above. These results indicate that (i) plus-strand primer cleavage and translocation are distinct steps that can be dissociated by mutation, (ii) lesions in sequences not included in the primer can severely inhibit primer translocation, and (iii) elongation of such untranslocated primers is responsible for the variable quantities of linear DNA that are found in all hepadnaviral stocks.
Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein
Authors: Homann H E , Willenbrink W , Buchholz C J , Neubert W J
Published: January 2020

Proteins from Sendai virus particles and from infected cells were analyzed in a protein-blotting protein-overlay assay for their interaction with in vitro-synthesized, [35S]methionine-labeled viral proteins NP, P, and M. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto polyvinylidene difluoride membranes, and renaturation, the immobilized proteins were found to interact specifically with radiolabeled proteins. NP proteins from virus particles and from infected cells retained 35S-P protein equally well. Conversely, P protein from virus particles and from infected cells retained 35S-NP protein. 35S-M protein was retained mainly by NP protein but also by several cellular proteins. To determine the domains on NP protein required for binding to immobilized P protein, a series of truncated and internally deleted 35S-NP proteins was constructed. The only deletion that did not affect binding resides between residues 426 and 497. The carboxyl-terminal 27 residues (positions 498 to 524) contribute significantly to the binding affinity. Removal of 20 residues (positions 225 to 244) in the hydrophobic middle part of NP protein completely abolished its binding to P protein.
Production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of interleukin-6 (IL-6) but not IL-4, IL-5, and gamma interferon
Authors: Moskophidis D , Frei K , Löhler J , Fontana A , Zinkernagel R M
Published: January 2020

The activities of cytokines were determined in cerebrospinal fluid (CSF) and serum of mice persistently or intracerebrally acutely infected with lymphocytic choriomeningitis (LCM) virus (LCMV). In contrast to CBA/J (LCMV carrier) mice that responded with low levels of LCMV-specific antibody, high-responder NMRI (carrier) mice showed antibody production by B cells outside of lymphoid organs. The B cells that had infiltrated the brains of LCMV carrier mice exhibited no preferential immunoglobulin isotype or subtype virus-specific antibody production. Phenotypic analysis of the brain infiltrates in virus carrier mice revealed dominance of CD4+ T cells in contrast to virtual absence of CD4+ and dominance of CD8+ in mice with acute LCM. In NMRI but not in CBA/J carrier mice, significant concentrations of interleukin-6 (IL-6) were detected in CSF and serum; IL-2, IL-4, IL-5, granulocyte-macrophage CSF (GM-CSF), and gamma interferon (IFN-gamma) were not elevated. In contrast, during acute, lethal LCM, IL-6 and IFN-gamma were found at high concentrations, and IL-4, IL-5, and GM-CSF were detectable in CSF and serum, but virus-specific antibody-producing cells were not (yet) detectable in the brain. Thus, distinct cytokine patterns are found in acute versus chronic LCMV infection of the brain: in LCM carrier mice, local random-class immunoglobulin production correlated with the absence of IL-2, IL-4, IL-5, and IFN-gamma but active secretion of IL-6.
Extended life span of human endometrial stromal cells transfected with cloned origin-defective, temperature-sensitive simian virus 40
Authors: Rinehart C A , Haskill J S , Morris J S , Butler T D , Kaufman D G
Published: January 2020

Human endometrial stromal cells transfected with an origin-defective, temperature-sensitive simian virus 40 recombinant plasmid are dependent on T-antigen function for proliferation and at the permissive temperature have an extended life span in culture. Southern blot analysis indicates that the transfected gene is present in low copy number, possibly at a single integration site. Normal stromal cells are capable of 10 to 20 population doublings in culture. Transfected cultures have been carried at the permissive temperature to 80 population doublings before crisis. In the multistep model of malignant transformation of human cells, these cells represent one of the earliest stages: extended but finite life span. We have used these cells to investigate alterations in signal transduction that may be responsible for this early stage of transformation caused by the large T antigen. Temperature shift experiments indicate that the expression of ornithine decarboxylase (ODC) but not of c-fos is altered by the large T antigen. Induction of c-fos by serum or 12-O-tetradecanoylphorbol-13-acetate is independent of temperature. However, in the transfected cells, the induction of ODC by asparagine or serum is greatly enhanced at the permissive temperature. This result indicates that the large T antigen acts downstream of c-fos but upstream of ODC expression in the signal-transducing cascade.
Human T-cell leukemia-lymphoma virus type I (HTLV-I) expression in fresh peripheral blood mononuclear cells from patients with tropical spastic paraparesis/HTLV-I-associated myelopathy
Authors: Gessain A , Louie A , Gout O , Gallo R C , Franchini G
Published: January 2020

Tropical spastic paraparesis/human T-cell leukemia-lymphoma virus type I (HTLV-I)-associated myelopathy (TSP/HAM) is a chronic neurological illness epidemiologically associated with HTLV-I infection. We investigated the role of HTLV-I in the pathogenesis of this disease by studying viral expression in fresh uncultured peripheral blood mononuclear cells (PBMCs) of six patients of Caribbean origin with TSP/HAM. The PBMC genomic DNA of all the patients studied carried HTLV-I provirus, but viral expression was not detected by Northern (RNA) blot analysis of total cellular PBMC RNA. When the reverse transcriptase polymerase chain reaction technique was used with primers specific for the tax-rex mRNA, all of the samples were positive for this viral mRNA species, regardless of the duration of the illness (range, 2 to 13 years). The splice junctions for the tax-rex mRNA described in cases of HTLV-I-induced adult T-cell leukemia (position 5183 of the envelope and position 7302 of the pX region) were identical in three TSP/HAM cases studied. To ascertain whether viral expression occurred at a low level in many cells or at a high level in a few permissive cells, we performed in situ hybridization on fresh PBMCs from two patients (2 and 7 years after clinical diagnosis), seeking HTLV-I RNA sequences. Our finding indicated that in vivo HTLV-I expression occurred at a high level in a few cells (1 of every 5,000 PBMCs) in both cases studied. The fact that cells of all six patients with TSP/HAM were positive for viral expression, regardless of the time lag from diagnosis, suggests that persistent expression of a viral product(s) may be pivotal in the pathogenesis of TSP/HAM.
Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived
Authors: Connors M , Collins P L , Firestone C Y , Murphy B R
Published: January 2020

The ability of recombinant vaccinia viruses that separately encoded 9 of the 10 known respiratory syncytial virus (RSV) proteins to induce resistance to RSV challenge was studied in BALB/c mice. Resistance was examined at two intervals following vaccination to examine early (day 9) as well as late (day 28) immunity. BALB/c mice were inoculated simultaneously by the intranasal and intraperitoneal routes with a recombinant vaccinia virus encoding one of the following RSV proteins: F, G, N, P, SH, M, 1B, 1C, or M2 (22K). A parainfluenza virus type 3 HN protein recombinant (Vac-HN) served as a negative control. One half of the mice were challenged with RSV intranasally on day 9, and the remaining animals were challenged on day 28 postvaccination. Mice previously immunized by infection with RSV, Vac-F, or Vac-G were completely or almost completely resistant to RSV challenge on both days. In contrast, immunization with Vac-HN, -P, -SH, -M, -1B, or -1C did not induce detectable resistance to RSV challenge. Mice previously infected with Vac-M2 or Vac-N exhibited significant but not complete resistance on day 9. However, in both cases resistance had largely waned by day 28 and was detectable only in mice immunized with Vac-M2. These results demonstrate that F and G proteins expressed by recombinant vaccinia viruses are the most effective RSV protective antigens. This study also suggests that RSV vaccines need only contain the F and G glycoproteins, because the immunity conferred by the other proteins is less effective and appears to wane rapidly with time.
Structure and expression of murine cytomegalovirus immediate-early gene 2
Authors: Messerle M , Keil G M , Koszinowski U H
Published: January 2020

The immediate-early gene ie2 of murine cytomegalovirus was characterized. The 1.75-kb ie2 transcript is spliced from three exons, of 78, 124, and 1,283 nucleotides, which are separated by introns of 1,245 and 364 nucleotides. An ATG codon located in the third exon leads into an open reading frame of 391 codons. Immediate-early expression of the predicted polypeptide was confirmed by immunoprecipitation of a 43-kDa protein by using an antiserum raised against a bacterial fusion protein. The predicted IE2 amino acid sequence has regions with similarity to amino acid sequences of members of the human cytomegalovirus US22 family.
Cloning and expression of the vesicular stomatitis virus phosphoprotein gene in Escherichia coli: analysis of phosphorylation status versus transcriptional activity
Authors: Barik S , Banerjee A K
Published: January 2020

The phosphoprotein (P, previously known as NS) genes of vesicular stomatitis virus serotypes New Jersey and Indiana have been cloned in the Escherichia coli expression vector pET-3a. Transcription of P genes in these clones initiated from a phage T7 RNA polymerase promoter, whereas translation was driven by the Shine-Dalgarno sequence and the initiator AUG codon of the T7 gene 10 message. The clones were introduced into an appropriate E. coli strain in which T7 RNA polymerase was expressed under the control of the lac promoter. Under optimal conditions of induction with isopropylthiogalactopyranoside, P protein made in these bacterial strains constituted 5 to 20% of total cellular protein. P protein expressed in bacteria was unphosphorylated and transcriptionally active in an in vitro reconstitution assay with viral L protein and an N-RNA template. However, the P protein was phosphorylated in vitro by the kinase activities associated with L and the N-RNA template.
Human immunodeficiency virus type 1 Tat does not transactivate mature trans-acting responsive region RNA species in the nucleus or cytoplasm of primate cells
Authors: Chin D J , Selby M J , Peterlin B M
Published: January 2020

Human immunodeficiency virus (HIV)-encoded transactivator Tat is essential for viral gene expression and replication. By interacting with a nascent RNA stem-loop called the trans-acting responsive region (TAR). Tat increases rates of initiation and/or elongation of HIV transcription. Several reports have also suggested that Tat has additional effects on mature HIV RNA species including modification of primary transcripts in the nucleus and their increased translation in the cytoplasm. These posttranscriptional effects are most pronounced in the Xenopus oocyte. To investigate directly whether Tat has similar effects on viral transcripts in cells that are permissive for HIV replication, we cotransfected and microinjected human and monkey cells with Tat and TAR in the form of DNA or RNA. Whereas Tat transactivated TAR DNA targets, it did not transactivate TAR RNA targets in the nucleus of microinjected cells or in the cytoplasm of transfected cells. We conclude that in cells permissive for viral replication, Tat exerts its effect primarily at the level of HIV transcription.
Activator-dependent and activator-independent defective recombinant retroviruses from bovine leukemia virus
Authors: Milan D , Nicolas J F
Published: January 2020

The replication-competent bovine leukemia virus (BLV) has been modified for use as a vector for foreign genes. The gag, pol, env, and pX regions of the virus were replaced by an exogenous nuclear location signal LacZ (nlsLacZ) or SVnlsLacZ gene. Transfection of the ovine cell line FLK-BLV, which expresses all BLV proteins from a wild-type provirus, with this viral DNA resulted in a viral titer of 10(4) CFU/ml. The inclusion of a large portion of the gag region did not significantly increase the titer. Both activator-dependent and activator-independent retroviruses were constructed. In activator-dependent vectors, the expression of the insert was dependent on the presence of the Tax protein, which activated the BLV long terminal repeat. In activator-independent vectors, the expression of the insert was constitutive because of the presence of an internal promoter. Infections with the recombinant retrovirus were inhibited by specific neutralizing antibodies. The structure of the transduced genetic material was not rearranged. BLV vectors encoding a reporter nlsLacZ gene, the product of which can be detected in single cells, greatly simplified studies of their biological properties. Determination of the host range of BLV vectors established that BLV-based recombinant retroviruses are effective in the transduction of genes in a variety of species and cell types.
Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction
Authors: Borroto-Esoda K , Boone L R
Published: January 2020

The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus-strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic bands were observed in some cases, and both the size and proportion varied with reaction conditions. Conditions established for full-length EIAV DNA synthesis also allowed full-genome-length human immunodeficiency virus type 1 DNA synthesis. The human immunodeficiency virus type 1 DNA product contained a greater proportion of reverse transcripts that were shorter than the complete virus genome. Also in contrast to EIAV, the endogenous synthesis of high-molecular-weight human immunodeficiency virus type 1 DNA was drastically reduced at Nonidet P-40 concentrations above 0.02%. These results indicated that a detergent-stable core is not a property shared by all lentiviruses. The EIAV virion synthetic machinery is unusually stable and provides a convenient system for further in vitro study of reverse transcription.
Dissociation and reassociation of oligomeric viral glycoprotein subunits in the endoplasmic reticulum
Authors: Zagouras P , Ruusala A , Rose J K
Published: January 2020

The vesicular stomatitis virus (VSV) glycoprotein (G) forms noncovalently linked trimers in the endoplasmic reticulum (ER) prior to transport to the cell surface. Here we examined the formation of heterotrimers between wild-type and mutant subunits that were retained in the ER by C-terminal retention signals. When G protein was coexpressed with mutant subunits that formed trimers at the wild-type rate and were transported from the ER at the wild-type rate, heterotrimers were readily detected. In contrast, when G protein was coexpressed with mutant subunits that formed trimers at the wild-type rate, but were retained in the ER, heterotrimers were not detected unless transport of the wild-type molecules from the ER was blocked. After removal of transport block, the heterotrimers then dissociated and reassorted to homotrimers of the mutant protein that were retained in the ER and wild-type trimers that were transported to the cell surface. These and other results presented here indicate that there is an equilibrium between G protein trimers and monomers in vivo, at least in the ER. This equilibrium may function to allow escape of wild-type subunits from aberrant retained subunits.