bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that developmentally regulated control of gene expression by a parvoviral allotropic determinant is particle mediated
Authors: Gardiner E M , Tattersall P
Published: January 2020

An infectious molecular clone of the immunosuppressive strain of the autonomous parvovirus minute virus of mice [MVM(i)] was constructed deriving left-hand terminal sequences from a rare encapsidated plus strand. Progeny virus was shown to package the same proportions of plus and minus strands as did authentic MVM(i) virions. Rescue of virus from this clone also resulted in the repair of a 21-base truncation at the junction between the right-hand end of the viral insert and the vector and generated the same heterogeneous 5' end as is present in standard MVM(i) DNA. Progeny virus rescued by transfection of this clone into mouse cell lines displayed the lymphotropic phenotype characteristic of the parental MVM(i) virus from which it was derived. However, analysis of viral RNA from transfected mouse fibroblasts revealed that the MVM(i) and MVM(p) genomic clones are transcribed at the same low level. Furthermore, transfected fibroblasts yielded similar numbers of infectious centers regardless of which MVM clone was introduced. These results contrast markedly with the different infectivities of MVM(i) and MVM(p) particles and with the observation that viral transcription in fibroblasts productively infected with MVM(p) virions is 100-fold greater than that seen in the restrictive MVM(i) particle-mediated infection. These results suggest that the developmentally regulated intracellular factors controlling host cell susceptibility at the level of viral transcription interact with a component of the incoming viral capsid, rather than with a sequence within the viral DNA.
Clearance of lymphocytic choriomeningitis virus in antibody- and B-cell-deprived mice
Authors: Cerny A , Sutter S , Bazin H , Hengartner H , Zinkernagel R M
Published: January 2020

The role of antibody in immune recovery from infection with lymphocytic choriomeningitis virus (LCMV) strain WE was evaluated in B-cell-depleted mice. Mice were treated from birth with either affinity-purified rabbit anti-mouse immunoglobulin M (IgM), normal rabbit immunoglobulin, or, alternatively, an affinity-purified monoclonal rat anti-mouse IgM antibody (LO-MM-9); untreated mice served as controls. B-cell depletion was considered complete in specifically treated mice according to the following criteria: absence of a significant response to the B-cell mitogen lipopolysaccharide, absence of B cells expressing immunoglobulin on their surfaces, absence of detectable IgM or IgG in serum, and presence in the serum of free anti-IgM antibodies. In organs of mu-suppressed BALB/c mice, LCMV-WE replicated, dependent upon organ, at the same rate or more rapidly and, in general, to higher titers than in normal rabbit immunoglobulin-treated mice; untreated mice eliminated the virus most rapidly and showed lower virus titers. In addition, LCMV-primed control mice cleared a second LCMV challenge very rapidly and contained no virus by day 3, whereas mu-suppressed mice had virus in their blood and organs (except the spleen) up to days 3 to 6. The observed effects of anti-mu treatment may reflect the action of neutralizing antibodies (which so far have been difficult to demonstrate in vivo) or other antibody-dependent antiviral mechanisms which, together with T cells, efficiently control LCMV clearance.
Standardized and simplified nomenclature for proteins common to all retroviruses
Authors: Leis J , Baltimore D , Bishop J M , Coffin J , Fleissner E , Goff S P , Oroszlan S , Robinson H , Skalka A M , Temin H M
Published: January 2020

We propose a revised standardized nomenclature for the proteins common to all retroviruses on the basis of biological function, enzymatic activity, and/or virion location data. (We do not discuss proteins specific for subfamilies or only some retroviruses.)
Evidence for a complex structure of neutralization antigenic site I of poliovirus type 1 Mahoney
Authors: Wiegers K , Uhlig H , Dernick R
Published: January 2020

We have selected neutralization escape mutants by using a monoclonal antibody (nt-MAb) against a sequential epitope between amino acids 93 through 104 (neutralization antigenic site I) of poliovirus type 1 Mahoney. The majority of mutants were also resistant against five strain-specific nt-MAbs which recognized conformation-dependent epitopes, suggesting that the neutralization antigenic site I must be involved in the formation of such epitopes. An analysis of all mutants by the binding of nt-MAbs and by isoelectric focusing of VP1 allowed discrimination of five classes of mutants. Sequence analysis of mutant RNAs revealed point mutations and deletions in the antibody-binding site.
Monoclonal antibodies define a domain on herpes simplex virus glycoprotein B involved in virus penetration
Authors: Highlander S L , Cai W H , Person S , Levine M , Glorioso J C
Published: January 2020

In an earlier report (S.D. Marlin, S.L. Highlander, T.C. Holland, M. Levine, and J.C. Glorioso, J. Virol. 59: 142-153), we described the production and use of complement-dependent virus-neutralizing monoclonal antibodies (MAbs) and MAb-resistant (mar) mutants to identify five antigenic sites (I to V) on herpes simplex virus type 1 glycoprotein B (gB). In the present study, the mechanism of virus neutralization was determined for a MAb specific for site III (B4), the only site recognized by MAbs which exhibited complement-independent virus-neutralizing ability. This antibody had no detectable effect on virus attachment but neutralized viruses after adsorption to cell monolayers. These findings implied that the mechanism of B4 neutralization involved blocking of virus penetration. The remaining antibodies, which recognized sites I, II, and IV, required active complement for effective neutralization. These were further studied for their ability to impede virus infectivity in the absence of complement. Antibodies to sites I (B1 and B3) and IV (B6) slowed the rate at which viruses penetrated cell surfaces, supporting the conclusion that antibody binding to gB can inhibit penetration by a virus. The data suggest that MAbs can interfere with penetration by a virus by binding to a domain within gB which is involved in this process. In another assay of virus infection, MAb B6 significantly reduced plaque development, indicating that antibody binding to gB expressed on infected-cell surfaces can also interfere with the ability of a virus to spread from cell to cell. In contrast to these results, antibodies to site II (B2 and B5) had no effect on virus infectivity; this suggests that they recognized structures which do not play a direct role in the infectious process. To localize regions of gB involved in these phenomena, antibody-binding sites were operationally mapped by radioimmunoprecipitation of a panel of truncated gB molecules produced in transient-expression assays. Residues critical to recognition by antibodies which affect penetration by a virus (sites I, III, and IV) mapped to a region of the molecule (amino acid residues 241 to 441) which is centrally located within the external domain. Antibodies which had no effect on penetration (site II) recognized sequences distal to this region (residues 596 to 737) near the transmembrane domain. The data suggest that these gB-specific MAbs recognize two major antigenic sites which reside in physically distinct components of the external domain of gB.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of primary human keratinocytes transformed by human papillomavirus type 18
Authors: Kaur P , McDougall J K
Published: January 2020

Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. One cell strain (FE-A), which exhibits an extended life span, is currently in its 30th passage. In comparison, control cultures can only be maintained up to the seventh passage. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes (M. Steinberg and V. Defendi, J. Cell Physiol. 123:117-125, 1985). FE-A cells were also found to be defective in their response to terminal differentiation stimuli. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.
The contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein D
Authors: Wilcox W C , Long D , Sodora D L , Eisenberg R J , Cohen G H
Published: January 2020

Glycoprotein D (gD) is an envelope component of herpes simplex virus types 1 (gD-1) and 2 (gD-2). The gD-1 polypeptide contains seven cysteine residues among its 369 amino acids; six are located on the N-terminal or luminal portion of the glycoprotein, and a seventh is located in the transmembrane region. Previous studies used a panel of monoclonal antibodies (MAbs) to define gD epitopes as continuous or discontinuous. Purified gD, denatured by reduction and alkylation, loses discontinuous epitopes, whereas continuous epitopes are retained. The contribution of disulfide bonds to maintenance of discontinuous epitopes is, therefore, significant. In the present study, our objective was to determine the contribution of individual cysteine residues to folding of gD-1 into its native conformation. Site-directed oligonucleotide mutagenesis was used to create seven mutants, each with a serine residue replacing a cysteine. The mutated genes were cloned into a eucaryotic expression vector and transfected into COS-1 cells, and the proteins were separated by nondenaturing polyacrylamide gel electrophoresis, followed by immunoblotting. Replacement of cysteine 7 (residue 333) had only a minimal effect on the antigenic properties of gD-1. In contrast, replacement of any one of the other six cysteine residues resulted in either a major reduction or a complete loss of binding of those MAbs that recognize discontinuous epitopes, with no effect on the binding of MAbs which recognize continuous epitopes. These mutations also had profound effects on the extent of oligosaccharide processing of gD-1. This was determined by digestion of the expressed proteins with various endoglycosidases, followed by electrophoresis and Western blotting (immunoblotting) to observe any mobility changes. Three mutant gD proteins which did not express discontinuous epitopes contained only high-mannose-type oligosaccharides, suggesting that processing had not proceeded beyond the precursor stage. Two mutant forms of gD exhibited reduced binding of MAbs to discontinuous epitopes. A small proportion of the molecules which accumulated at 48 h posttransfection contained complex oligosaccharides. One mutant exhibited reduced binding of MAbs to discontinuous epitopes, but was present at 48 h posttransfection only in the precursor form. The cysteine 7 mutant was processed to the same extent as wild-type gD. We conclude that the first six cysteine residues are critical to the correct folding, antigenic structure, and processing of gD-1, and we speculate that they form three disulfide-bonded pairs.
Cell lines containing varicella-zoster virus open reading frame 62 and expressing the "IE" 175 protein complement ICP4 mutants of herpes simplex virus type 1
Authors: Felser J M , Kinchington P R , Inchauspe G , Straus S E , Ostrove J M
Published: January 2020

Vero cells were cotransfected with pSV2neo and a recombinant plasmid containing the varicella-zoster virus (VZV) open reading frame 62 (ORF62). Three neomycin-resistant cell lines were isolated and shown to complement two different ICP4 mutants (tsB21 and d120) of herpes simplex virus (HSV) type 1 (HSV-1). VZV-specific RNA could not be detected in these cell lines, but following infection with tsB21, a 4.3-kilobase VZV transcript was detected. This RNA increased in quantity when cells were infected in the presence of cycloheximide. A VZV-specific protein of 175 kilodaltons was detected in extracts of all three cell lines following infection with wild-type HSV-1 but not in uninfected cells. That VZV RNA and protein were detected only in HSV-1 infected cells suggests that a component of the HSV virion activates the expression of VZV ORF62. The increase in RNA expression seen in the presence of cycloheximide indicates that the protein encoded by VZV ORF62, "IE"175, may be autoregulatory. These data provide further evidence that VZV "IE"175 is the functional analog of the HSV ICP4.
Distribution of a macaque immunosuppressive type D retrovirus in neural, lymphoid, and salivary tissues
Authors: Lackner A A , Rodriguez M H , Bush C E , Munn R J , Kwang H S , Moore P F , Osborn K G , Marx P A , Gardner M B , Lowenstine L J
Published: January 2020

Simian acquired immune deficiency syndrome (SAIDS) in rhesus macaques (Macaca mulatta) at the California Primate Research Center is caused by a type D retrovirus designated SAIDS retrovirus serotype 1 (SRV-1). This syndrome is characterized by profound immunosuppression and death associated with opportunistic infections. Neurologic signs and lesions have not been described as part of this syndrome. The distribution of SRV-1 in the salivary glands, lymph nodes, spleens, thymuses, and brains of eight virus-infected rhesus macaques was examined by immunohistochemistry. Electron microscopy, in situ RNA hybridization, and Southern blot hybridization were also performed on selected tissues to detect viral particles, RNA, and DNA, respectively. In seven of eight SRV-1-infected animals, the transmembrane envelope glycoprotein (gp20) of SRV-1 was present in three or more tissues, but never in the brain. In the remaining animal, no viral antigen was detected in any tissue. In this same group of animals, viral nucleic acid was detected in the lymph nodes of six of six animals by Southern blot hybridization, in the salivary glands of two of five animals by both Southern blot and in situ hybridizations, and, surprisingly, in the brains of three of three animals by Southern blot and of three of five animals by in situ hybridization, including the one animal in which viral gp20 was undetectable. None of these animals had neurologic signs or lesions. The detection of viral nucleic acid in the absence of viral antigen in the brain suggests latent SRV-1 infection of the central nervous system.
Isolation of a transformation-defective mutant of the McDonough strain of feline sarcoma virus exhibiting tyrosine kinase activity in vitro but not in vivo
Authors: Tamura T , Hennig D , Grell M , Niemann H , Boschek B
Published: January 2020

NRK cells transformed by the McDonough strain of feline sarcoma virus (SM-FeSV) were mutagenized by the use of 5'-azacytidine. Four cell lines expressing different transformation-defective phenotypes were isolated. Superinfection of these cell lines with simian sarcoma-associated virus (SSAV) led in three instances to the recovery of transforming virus particles carrying an intact fms gene. A nonconditional transformation-defective virus, designated td26-SM-FeSV (SSAV), was isolated from one of the cell lines. NRK cells infected with this mutant contained actin cables and fibronectin networks and exhibited normal cell morphology. Such cells formed only small colonies in soft agar and exhibited a mitogenic activity similar to that of noninfected cells. Cells infected with td26-SM-FeSV (SSAV) synthesized a gag-fms fusion glycoprotein (gp180gag-fms). This polypeptide was processed in the normal manner into the intracellular gp120v-fms and a transformation-defective gp140td-v-fms which was expressed at the surface of infected cells. This species had an increased electrophoretic mobility on polyacrylamide gels compared with the molecule from wild-type virus.gp140td-v-fms had endo-beta-N-acetylglucosaminidase H-resistant carbohydrate side chains. No tyrosine kinase activity was detectable in vivo in td26-SM-FeSV (SSAV)-infected cells even when the cells were treated with sodium orthovanadate. In vitro, fms molecules from td26-SM-FeSV (SSAV)-infected cells exhibited tyrosine kinase activity as determined by autophosphorylation and phosphorylation of exogenous (poly)Glu-Tyr. At low ATP concentrations (less than 5 microM) this in vitro tyrosine kinase activity was significantly reduced compared with that of the wild-type counterpart.
A sixth Epstein-Barr virus nuclear protein (EBNA3B) is expressed in latently infected growth-transformed lymphocytes
Authors: Petti L , Kieff E
Published: January 2020

In the Epstein-Barr virus BamHI E genomic fragment, there are three distantly homologous long open reading frames, BERF1, BERF2b, and BERF4, each of which is preceded by a short open reading frame. The most leftward and most rightward short and long open reading frame pairs encode 145- and 155-kilodalton proteins in latently infected cells (EBNA3A and EBNA3C, respectively). In this report, we demonstrate that the middle long open reading frame, BERF2b, encodes part of a 165-kilodalton nuclear protein in every latently infected cell. Therefore, this protein is designated EBNA3B.
A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen
Authors: Hardwick J M , Lieberman P M , Hayward S D
Published: January 2020

Several Epstein-Barr virus (EBV) early promoters respond to a new EBV transactivator encoded by BRLF1, designated R. Transactivation was measured in chloramphenicol acetyltransferase assays on Raji, BHK, and Vero cells that were cotransfected with the transactivator and target promoters linked to the cat gene. The divergent promoter of BamHI-H was particularly responsive to R transactivation. This large promoter region consists of a leftward TATA box for the NotI repeat gene (BHLF1) and a probable rightward TATA box for the EA-R gene (BHRF1) separated by 940 base pairs of unusual sequence complexity. Sequences within this divergent promoter region appear to confer inducibility by EBV transactivators R and Z (BZLF1). The Z transactivator stimulated expression in both the leftward and rightward directions, and R stimulated expression primarily in the rightward direction, but the MS transactivator (BMLF1) had no activity in either direction. The adenovirus E3 promoter also responded to the R transactivator, but several other herpesvirus and human promoters were nonresponsive. When the divergent promoter was linked to the EA-R gene as it is in the EBV genome, the R and Z transactivators also induced the expression of EA-R in cotransfected cells. This cytoplasmic early antigen is encoded by BHRF1 and may be anchored in intracellular membranes by a carboxy-terminal transmembrane region.
Genetic evidence that the avian retrovirus DNA endonuclease domain of pol is necessary for viral integration
Authors: Quinn T P , Grandgenett D P
Published: January 2020

We used in vitro mutagenesis in the 3' region of the avian retrovirus polymerase (pol) gene to genetically define the role of the DNA endonuclease domain. In-frame insertional mutations, which were dispersed throughout the 5' region of pp32, produced a series of five replication-deficient mutants. In contrast, a single point mutant (Ala----Pro) located 48 amino acids from the NH2 terminus of pp32 exhibited a delayed replication phenotype. Molecular analysis of this mutant demonstrated that upon infection it was capable of synthesizing both linear and circular species of unintegrated viral DNA. The levels of unintegrated viral DNA present in cells infected with the mutant virus were several times greater than wild-type levels. Quantitation of the amount of integrated viral genomes demonstrated that the mutant virus integrated viral DNA one-fifth as efficiently as wild-type virus. This single point mutation in the NH2 terminus of pp32 prevented efficient integration of viral DNA, with no apparent effect on viral DNA synthesis per se. Thus, the DNA endonuclease domain has been genetically defined as necessary for avian retrovirus integration.
Cell surface expression and orientation in membranes of the 44-amino-acid SH protein of simian virus 5
Authors: Hiebert S W , Richardson C D , Lamb R A
Published: January 2020

Antiserum was raised against a synthetic peptide containing the N-terminal hydrophilic domain of the small hydrophobic protein (SH) of simian virus 5 (SV5) and used to characterize properties of the SH protein. SH demonstrated properties of an integral membrane protein. Indirect immunofluorescence experiments showed that the protein is involved in the exocytotic pathway, and isolation of plasma membranes from SV5-infected cells showed an enrichment of SH, indicating that SH is transported to the infected-cell surface. Biochemical analysis of the orientation of SH in membranes by proteolysis of intact SV5-infected cell surfaces and intracellular microsomal vesicles indicated that SH is oriented in membranes with its N-terminal hydrophilic domain exposed on the cytoplasmic face of the plasma membrane and the C terminus of approximately five amino acid residues exposed at the cell surface. These data are discussed with respect to positive-acting signals being necessary in the ectodomain of SH for cell surface expression.
Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli
Authors: Terry R , Soltis D A , Katzman M , Cobrinik D , Leis J , Skalka A M
Published: January 2020

The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.
Structure and complete nucleotide sequence of the Marek's disease herpesvirus gp57-65 gene
Authors: Coussens P M , Velicer L F
Published: January 2020

The gene encoding the precursor polypeptide of the Marek's disease herpesvirus (MDHV) secretory glycoprotein gp57-65 (formerly identified as A antigen) has been sequenced. Previous results had localized the gene to a 4.6-kilobase (kb) segment of the BamHI B fragment in the unique long region of the MDHV genome. S1 nuclease protection experiments were used to more precisely locate the 5' initiation and approximate 3' termination points of the approximately 1.8-kb MDHV gp57-65 mRNA within this segment. These results indicated that the entire MDHV gp57-65 coding sequence is contained within a 2.35-kb PvuII-EcoRI fragment, with the direction of transcription from PvuII to EcoRI (5' to 3'). Nucleotide sequence analysis of this region revealed a single open reading frame of 1,515 base pairs. The MDHV gp57-65 coding sequence has an overall guanosine-plus-cytosine content of 41%. Translation of the single open reading frame would produce a polypeptide of 505 amino acids, with a calculated molecular weight of 56,805. The putative gp57-65 precursor polypeptide contains features common to many glycoproteins. These include a hydrophobic amino-terminal region (amino acids 1 to 27) that may function as a signal peptide and nine potential N-linked glycosylation sites (Asn-X-Ser/Thr). These two features, predicted from nucleotide sequence data, are consistent with the published data showing that gp57-65 has a signal peptide and N-linked glycosylation (R. J. Isfort, R. A. Stringer, H.-J. Kung, and L. F. Velicer, J. Virol. 57:464-474, 1986). The predicted sequence indicates that overall the polypeptide is relatively hydrophobic, with a possible 18-residue carboxyl-terminal membrane anchor sequence. This sequence appears to be less prominent than those commonly found in integral membrane glycoproteins. The lack of a strong hydrophobic anchor sequence may help to explain the predominantly secretory nature of MDHV gp57-65.
Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA
Authors: French R , Ahlquist P
Published: January 2020

Expression of brome mosaic virus (BMV) coat protein and internal genes of many other positive-strand RNA viruses requires initiation of subgenomic mRNA synthesis from specific internal sites on minus-strand genomic RNA templates. Biologically active viral cDNA clones were used to investigate the sequences controlling production of BMV subgenomic RNA in vivo. Suitable duplications directed production of specifically initiated, capped subgenomic RNAs from new sites in the BMV genome. Previously implicated promoter sequences extending 20 bases upstream (-20) and 16 bases downstream (+16) of the subgenomic RNA initiation site directed only low-level synthesis. Subgenomic RNA production at normal levels required sequences extending to at least -74 but not beyond -95. Loss of an (rA)18 tract immediately upstream of the -20 to +16 "core promoter" particularly inhibited subgenomic RNA synthesis. The -38 to -95 region required for normal initiation levels contains repeats of sequence elements in the core promoter, and duplications creating additional upstream copies of these repeats stimulated subgenomic RNA synthesis above wild-type levels. At least four different subgenomic RNAs can be produced from a single BMV RNA3 derivative. For all derivatives producing more than one subgenomic RNA, a gradient of accumulation progressively favoring smaller subgenomic RNAs was seen.
Addition of substitution of simian virus 40 enhancer sequences into the Moloney murine leukemia virus (M-MuLV) long terminal repeat yields infectious M-MuLV with altered biological properties
Authors: Hanecak R , Pattengale P K , Fan H
Published: January 2020

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-cell lymphoma in mice. The enhancer sequences present within the M-MuLV long terminal repeat (LTR) region of the proviral genome have been shown to influence the disease specificity of the virus strongly. We examined the contribution of the M-MuLV enhancers to the transcriptional activity and pathogenesis of M-MuLV by constructing LTRs containing heterologous enhancer elements. The simian virus 40 enhancer region (72- and 21-base-pair repeats) was inserted into the U3 region (at -150 base pairs) of the M-MuLV LTR (Mo + SV) and also into a deleted form of the LTR which lacks the M-MuLV enhancer sequences (delta Mo + SV). These chimeric LTRs were used to generate infectious M-MuLVs by transfection of corresponding proviral plasmids into mouse fibroblasts. The relative infectivities of Mo + SV and delta Mo + SV recombinant viruses as determined by rat XC cell plaque assay and reverse transcriptase assay were 60 to 70% of wild-type M-MuLV levels. To study the pathogenicity of these two recombinant viruses, we inoculated newborn NIH Swiss mice with either Mo + SV or delta Mo + SV M-MuLV. Both viruses induced disease more slowly than M-MuLV, which induces disease 2 to 4 months postinoculation. Mo + SV M-MuLV-inoculated animals became moribund at 3 to 13 months postinoculation, whereas delta Mo + SV M-MuLV-inoculated animals became moribund at 6 to 24 months postinoculation. The tumors induced by the two viruses were characterized histologically and molecularly. Mo + SV M-MuLV-induced tumors were primarily T-cell-derived lymphoblastic lymphomas containing extensive rearrangements of the T-cell receptor beta gene. In contrast, delta Mo + SV M-MuLV induced pre-B- and B-cell lymphoblastic lymphomas, B-cell-derived follicular-center cell lymphomas, and acute myeloid leukemia. The delta Mo + SV tumor DNAs from B-lineage tumors were typically rearranged at the immunoglobulin gene loci and contained germ line configurations of the T-cell receptor beta gene. Southern blot hybridization confirmed that the tumor DNAs contained the predicted Mo + SV M-MuLV or delta Mo + SV M-MuLV provirus.
Subcellular localization of the human immunodeficiency virus trans-acting art gene product
Authors: Cullen B R , Hauber J , Campbell K , Sodroski J G , Haseltine W A , Rosen C A
Published: January 2020

The genome of the human immunodeficiency virus is distinguished from other animal retroviruses by the presence of several additional open reading frames. The protein product of one of these novel genes, which has been termed art or trs, is required for the expression of the virus structural genes but not for the expression of virus encoded regulatory proteins. Immunocytochemistry and subcellular fractionation demonstrate that the art protein is located predominantly in the nucleus. Therefore, any proposed mechanism for the function of art is likely to involve nuclear events.
A single 66-kilodalton polypeptide processed from the human immunodeficiency virus type 2 pol polyprotein in Escherichia coli displays reverse transcriptase activity
Authors: Le Grice S F , Zehnle R , Mous J
Published: January 2020

We have cloned the entire pol gene of human immunodeficiency virus type 2 into a high-level Escherichia coli expression system. Induction of cultures containing the recombinant plasmid, p2RTL1, leads to rapid accumulation of polypeptides of 66, 54, and 34 kilodaltons. We have designated the larger polypeptides reverse transcriptase, and we have designated the smaller polypeptide endonuclease. Purification of reverse transcriptase via ion-exchange and affinity chromatography yields the 66-kilodalton polypeptide, with which reverse transcriptase activity is associated. Purified enzyme furthermore displays a higher apparent molecular weight than its counterpart from human immunodeficiency virus type 1.
Antigenicity, function, and conformation of synthetic oligopeptides corresponding to amino-terminal sequences of wild-type and mutant matrix proteins of vesicular stomatitis virus
Authors: Shipley J B , Pal R , Wagner R R
Published: January 2020

The matrix (M) protein of vesicular stomatitis virus (VSV) has a major antigenic determinant (epitope 1) that maps to a region extending from amino acids 19 through 43 and transcription-inhibition activity that maps to the first 43 N-terminal amino acids (J.R. Ogden, R. Pal, and R. R. Wagner, J. Virol. 58:860-868, 1986). The M protein of temperature-sensitive mutant tsO23(III) is devoid of epitope 1 and transcription-inhibition activity and substitutes glutamic acid for glycine at amino acid 21 as well as having amino acid substitutions at positions 111 and 227 (K. Morita, R. Vanderoef, and J. Lenard, J. Virol. 61:256-263, 1987). We undertook to map more precisely epitope 1 and the transcription-inhibition region of VSV M protein by means of synthetic oligopeptides generated by an automated solid-phase protein synthesizer. A pentadecapeptide designated PI(wt, Gly21), corresponding to amino acids 17 to 31 of wild-type (wt) M protein, strongly bound monoclonal antibody MAb2 (directed to epitope 1); however, an analogous pentadecapeptide with glutamic acid substituted for glycine at position 21, designated PII(tsO23, Glu21), completely failed to recognize MAb2. Polyclonal antibody raised in rabbits immunized with PI(wt, Gly21) reacted strongly with wt M protein, the homologous pentadecapeptide, and, to a lesser extent, PII(tsO23, Glu21). Anti-PII(tsO23, Glu21) failed to recognize PI(wt, Gly21) or wt M protein. Anti-PI(wt, Gly21) competed efficiently for binding of MAb2 to wt M protein and was as effective as MAb2 in reversing inhibition of VSV transcription by wt M protein. Neither PI(wt, Gly21) nor PII(tsO23, Glu21) exhibited any ability to inhibit VSV transcription. However, a lysine-rich oligopeptide, PII(Met1-Leu20), corresponding to the first 20 N-terminal amino acids of wt M protein, and polylysine itself did inhibit VSV transcription, albeit much less efficiently than native wt M protein. Monospecific polyclonal antibody directed to the 20-mer oligopeptide PIII(Met1-Leu20) reversed transcription inhibition by M protein in a dose-dependent manner almost identical to that of anti-PI(wt, Gly21) and epitope 1-specific MAb2. Examination by circular dichroism spectropolarimetry revealed significant differences in the conformation of the two pentadecapeptides attributable to the Gly in equilibrium Glu amino acid substitution at position 21.
Inhibition of Epstein-Barr virus (EBV) release from P3HR-1 and B95-8 cell lines by monoclonal antibodies to EBV membrane antigen gp350/220
Authors: Sairenji T , Bertoni G , Medveczky M M , Medveczky P G , Nguyen Q V , Humphreys R E
Published: January 2020

Antibody-mediated inhibition of Epstein-Barr virus (EBV) release from the EBV-productive cell lines P3HR-1 and B95-8 was probed with two monoclonal antibodies (MAbs), 72A1 and 2L10, which immunoprecipitated the same EBV membrane antigen (MA) gp350/220 found with the 1B6 MAb with which inhibition of EBV release from P3HR-1 cells was first described. These three MAbs were not equivalent in either MA reactivities or functional effects, reflecting the variable expression of different epitopes of gp350/220. 1B6 recognized MA on P3HR-1 cells, which expressed predominately the gp220 form of MA. 1B6 did not recognize (or barely recognized) a determinant on B95-8 cells. MAbs 2L10 and 72A1 reacted as well with B95-8 cells as they did with P3HR-1 cells. MAbs 1B6 and 2L10 neutralized neither P3HR-1 nor B95-8 virus, but 72A1 neutralized both viruses. MAbs 1B6 and 72A1 inhibited P3HR-1 virus release, as measured by the assay for infectious virus and by DNA hybridization analysis of released virus, but 2L10 had no such activity. 72A1 (but not 1B6) inhibited release of EBV from B95-8 cells. These experiments pointed to the presence of three different epitopes on gp350/220, identified with the respective MAbs and having varying involvement in virus neutralization and virus release inhibition.
Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes
Authors: Boone L R , Glover P L , Innes C L , Niver L A , Bondurant M C , Yang W K
Published: January 2020

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.
Separate epitopes in the envelope of visna virus are responsible for fusion and neutralization: biological implications for anti-fusion antibodies in limiting virus replication
Authors: Crane S E , Clements J E , Narayan O
Published: January 2020

Visna virus is a lentivirus which causes fusion of infected cells in vitro. Two types of fusion occur. Fusion from without requires no viral replication and a relatively high multiplicity of infection; fusion from within results from the replication of virus in cells. By using fusion from without as an assay, the mechanism of fusion by visna virus was investigated. Immune sera which contained both anti-fusion and neutralizing antibodies interacted with the virus with rapid kinetics in blocking fusion but relatively slow kinetics in the virus neutralization assay. By using visna virus and an antigenic variant, the epitopes responsible for fusion and virus neutralization were shown to be different. Antigenic variation of visna virus resulted in alteration of the neutralization epitope and conservation of the fusion epitope. This suggested that there were two populations of antibodies and that the viral epitopes for fusion and neutralization were separate. These data suggest that visna virus is capable of infecting cells via two pathways: one via the fusion site and the other via the viral epitope which mediates neutralization.
Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells
Authors: Haddada H , Sogn J A , Coligan J E , Carbone M , Dixon K , Levine A S , Lewis A M
Published: January 2020

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.
Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions
Authors: Zebedee S L , Lamb R A
Published: January 2020

The influenza A virus M2 protein is an integral membrane protein of 97 amino acids that is expressed at the surface of infected cells with an extracellular N-terminal domain of 18 to 23 amino acid residues, an internal hydrophobic domain of approximately 19 residues, and a C-terminal cytoplasmic domain of 54 residues. To gain an understanding of the M2 protein function in the influenza virus replicative pathway, we produced and characterized a monoclonal antibody to M2. The antibody-binding site was located to the extracellular N terminus of M2 as shown by the loss of recognition after proteolysis at the infected-cell surface, which removes 18 N-terminal residues, and by the finding that the antibody recognizes M2 in cell surface fluorescence. The epitope was further defined to involve residues 11 and 14 by comparing the predicted amino acid sequences of M2 from several avian and human strains and the ability of the M2 protein to be recognized by the antibody. The M2-specific monoclonal antibody was used in a sensitive immunoblot assay to show that M2 protein could be detected in virion preparations. Quantitation of the amount of M2 associated with virions by two unrelated methods indicated that in the virion preparations used there are 14 to 68 molecules of M2 per virion. The monoclonal antibody, when included in a plaque assay overlay, considerably showed the growth of some influenza virus strains. This plaque size reduction is a specific effect for the M2 antibody as determined by an analysis of recombinants with defined genome composition and by the observation that competition by an N-terminal peptide prevents the antibody restriction of virus growth.
Antigenic sites on foot-and-mouth disease virus type A10
Authors: Thomas A A , Woortmeijer R J , Puijk W , Barteling S J
Published: January 2020

A set of monoclonal antibodies was used to isolate nonneutralizable foot-and-mouth disease virus variants, and the RNAs of the variants were sequenced. Cross-neutralization studies and mapping of the amino acid changes indicated two major antigenic sites. The first site was trypsin sensitive and included the VP1 140 to 160 sequence. The second site was trypsin insensitive and included mainly VP3 residues. Two minor sites were located near VP1 169 and on the C terminus of VP1. Comparison with poliovirus type 1 and human rhinovirus 14 showed a similarity in the immunogenicity of comparable sites on the viruses.
Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication
Authors: Dougherty J P , Temin H M
Published: January 2020

We recently described a protocol for determination of retrovirus mutation rates, that is, the mutation frequency in a single cycle of retrovirus replication (J.P. Dougherty and H.M. Temin, Mol. Cell. Biol. 6:4378-4395, 1987; J.P. Dougherty and H.M. Temin, p. 18-23, in J. H. Miller and M. P. Calos, ed., Gene Transfer Vectors for Mammalian Cells, 1987). We used this protocol to determine the mutation rates for defined mutations in a replicating retrovirus by using a spleen necrosis virus-based vector. We determined that the mutation rate for a single base pair substitution during replication of this avian retrovirus is 2 x 10(-5) per base pair per replication cycle and the insertion rate is 10(-7) per base pair per replication cycle. It will be possible to use this protocol to determine mutation rates for other retroviruses.
A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain
Authors: Kohara M , Abe S , Komatsu T , Tago K , Arita M , Nomoto A
Published: January 2020

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.
The gene encoding the nonstructural protein of B19 (human) parvovirus may be lethal in transfected cells
Authors: Ozawa K , Ayub J , Kajigaya S , Shimada T , Young N
Published: January 2020

The B19 parvovirus is a cause of bone marrow failure in humans. B19 is toxic to erythroid progenitor cells in vitro. Viral products possibly responsible for toxicity were explored by transfection of cloned B19 genome into HeLa cells. The nonstructural (NS) protein was detected in cells 30 h after transfection. Plasmids containing the B19 genome were transfected with selectable marker genes in stable transformation assays. Plasmids that contained the left side of the B19 genome, which encodes the NS protein of the virus, inhibited antibiotic-resistant colony formation. Transformation occurred when NS protein expression was blocked by mutation. Suppression of transformation by NS protein was not tissue specific, suggesting a role for NS protein in toxicity for nonpermissive cells without parvovirus replication or virion accumulation.