bluuebunny/crossref_metadata_2025 huggingface dataset:
The gIII glycoprotein of pseudorabies virus is involved in two distinct steps of virus attachment
Authors: Zsak L , Sugg N , Ben-Porat T , Robbins A K , Whealy M E , Enquist L W
Published: January 2020

The entry of herpesviruses into cells involves two distinct stages: attachment or adsorption to the cell surface followed by internalization. The virus envelope glycoproteins have been implicated in both stages. Pseudorabies virus attaches to cells by an early interaction that involves the viral glycoprotein gIII and a cellular heparinlike substance. We examined the role of gIII in the attachment process by analysis of a set of viruses carrying defined gIII mutations. The initial attachment of gIII mutants with an internal deletion of 134 amino acids (PrV2) to MDBK cells was indistinguishable from that of wild-type virus. The adsorption of these mutants was, however, much more sensitive than that of wild-type virus to competing heparin. Furthermore, while attachment of wild-type virus to MDBK cells led to a rapid loss of sensitivity to heparin, this was not the case with PrV2, which could be displaced from the cell surface by heparin after it had attached to the cells. We conclude that glycoprotein gIII is involved in two distinct steps of virus attachment and that the second of these steps but not the first is defective in PrV2.
Phenotype-specific phosphorylation of simian virus 40 tsA mutant large T antigens in tsA N-type and A-type transformants
Authors: Knippschild U , Kiefer J , Patschinsky T , Deppert W
Published: January 2020

To identify molecular differences between simian virus 40 (SV40) tsA58 mutant large tumor antigen (large T) in cells of tsA58 N-type transformants [FR(tsA58)A cells], which revert to the normal phenotype after the cells are shifted to the nonpermissive growth temperature, and mutant large T in tsA58 A-type transformants [FR(tsA58)57 cells], which maintain their transformed phenotype after the temperature shift, we asked whether the biological activity of these mutant large T antigens at the nonpermissive growth temperature might correlate with phosphorylation at specific sites. At the permissive growth temperature, the phosphorylation patterns of the mutant large T proteins in FR(tsA58)A (N-type) cells and in FR(tsA58)57 (A-type) cells were largely indistinguishable from that of wild-type large T in FR(wt648) cells. After a shift to the nonpermissive growth temperature, no significant changes in the phosphorylation patterns of wild-type large T in FR(wt648) or of mutant large T in FR(tsA58)57 (A-type) cells were observed. In contrast, the phosphorylation pattern of mutant large T in FR(tsA58)A (N-type) cells changed in a characteristic manner, leading to an apparent underphosphorylation at specific sites. Phosphorylation of the cellular protein p53 was analyzed in parallel. Characteristic differences in the phosphorylation pattern of p53 were observed when cells of N-type and A-type transformants were kept at 39 degrees C as opposed to 32 degrees C. However, these differences did not relate to the different phenotypes of FR(tsA58)A (N-type) and FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature. Our results, therefore, suggest that phosphorylation of large T at specific sites correlates with the transforming activity of tsA mutant large T in SV40 N-type and A-type transformants. This conclusion was substantiated by demonstrating that the biological properties as well as the phosphorylation patterns of SV40 tsA28 mutant large T in cells of SV40 tsA28 N-type and A-type transformants were similar to those in FR(tsA58)A (N-type) and in FR(tsA58)57 (A-type) cells, respectively. The phenotype-specific phosphorylation of tsA mutant large T in tsA A-type transformants probably is a cellular process induced during establishment of SV40 tsA A-type transformants, since tsA28 A-type transformant cells could be obtained by a large-T-dependent in vitro progression of cells of the tsA28 N-type transformant tsA28.3 (M. Osborn and K. Weber, J. Virol. 15:636-644, 1975).
Viral genetic determinants of T-cell killing and immunodeficiency disease induction by the feline leukemia virus FeLV-FAIDS
Authors: Donahue P R , Quackenbush S L , Gallo M V , deNoronha C M , Overbaugh J , Hoover E A , Mullins J I
Published: January 2020

Within the fatal immunodeficiency disease-inducing strain of feline leukemia virus, FeLV-FAIDS, are viruses which range in pathogenicity from minimally (clone 61E is the prototype) to acutely pathogenic, most of the latter of which are also replication defective (clone 61C is the prototype). Mixtures of 61E and 61C virus and chimeras generated between them, but not 61E alone, killed feline T cells. T-cell killing depended on changes within a 7-amino-acid region near the C terminus of the gp70 env gene or was achieved independently by changes within a 109-amino-acid region encompassing the N terminus of gp70. The carboxy-terminal change was also sufficient for induction of fatal immunodeficiency disease in cats. Other changes within the 61C gp70 gene enhanced T-cell killing, as did changes in the long terminal repeat, the latter of which also enhanced virus replication. T-cell killing correlated with high levels of intracellular unintegrated and proviral DNA, all of which were blocked by treatment of infected cells with sera from 61C-immune cats or with a neutralizing monoclonal antibody. These findings indicate that T-cell killing is a consequence of superinfection and that the mutations in env critical to pathogenicity of the immunosuppressive variant result in a failure to establish superinfection interference in infected cells.
Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1
Authors: Garfinkel D J , Hedge A M , Youngren S D , Copeland T D
Published: January 2020

Using antibodies directed against the TYB1 protein of the transpositionally competent retrotransposon Ty1-H3, we have identified three mature proteins of 23, 60, and 90 kDa and processing intermediates of 140 and 160 kDa that are derived from the 190-kDa TYA1-TYB1 polyprotein. Mature proteins and variable amounts of the precursors cofractionate with Ty viruslike particles. The map locations and precursor-product relationships of mature TYB1 polypeptides suggest that p23 is Ty1 protease, p90 is integrase, and p60 contains reverse transcriptase and RNase H. Immunoprecipitation and immunoblot analyses of Ty1 proteins show that p190 is cleaved to form p160. The p160 intermediate is cleaved to form p23 and p140, and p140 is cleaved to form p90 and p60. Processing of TYB1 proteins is dependent on Ty1 protease. Immunoblot analysis of TYB proteins from different Ty1 isolates reveal that correct processing of TYB1 proteins is a characteristic of functional Ty1 elements, whereas aberrant processing is a common defect found in transposition-incompetent elements.
Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein
Authors: Blasco R , Cole N B , Moss B
Published: January 2020

A 4,500-bp BamHI fragment, located within the HindIII A segment of the vaccinia virus genome, was found to contain eight potential coding regions for polypeptides of 78 to 346 amino acids. The open reading frames with 133, 346, and 125 codons were homologous to profilin (an actin-binding protein), 3-beta-hydroxysteroid dehydrogenase, and Cu-Zn superoxide dismutase, respectively. Sequence alignments indicated that the vaccinia virus and mammalian profilins were more closely related to each other than to known profilins of other eukaryotes. The expression and possible role of the profilin homolog in the virus replicative cycle were therefore investigated. Antibody raised to Escherichia coli expressed vaccinia virus profilin was used to demonstrate the synthesis of the 15-kDa polypeptide at late times after vaccinia virus infection of mammalian cells. The protein accumulated in the cytoplasm, but only trace amounts remained associated with highly purified virions. The isolation of vaccinia virus mutants (in strains WR and IHD-J), with nearly the entire profilin gene replaced by the E. coli gpt gene, indicated that the protein is not essential for infectivity. The characteristic vaccinia virus-induced changes in actin fibers, seen by fluorescence microscopy, occurred in cells infected with the mutant. Moreover, the virus-encoded profilin homolog was not required for actin-associated events, including intracellular virus movement to the periphery of the cell, formation of specialized microvilli, or release of mature virions, as shown by electron microscopy and yields of infectious intra- and extracellular virus.
The minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting
Authors: Willwand K , Hirt B
Published: January 2020

The terminal hairpin structures of the DNA of minute virus of mice (MVM) are essential for viral replication. Here we show that the hairpin 3' terminus of MVM replicative-form DNA binds specifically to empty MVM capsids. Binding of the same terminal DNA sequence in its linear double-stranded (extended) conformation was not observed. After heat denaturation and quick cooling of 3'-terminal extended-form fragments, not only the virion strand but also the complementary strand was found to bind to the capsid, presumably because each strand re-formed a similar hairpin structure. No binding affinity for the capsid was found to be associated with hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forming a binding site at the branch point of the two 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single base changes within this site did not affect the binding. In band shift experiments, specific binding to the T structure was demonstrated for VPI but not for VP2.
Characterization of the DNA polymerase gene of human herpesvirus 6
Authors: Teo I A , Griffin B E , Jones M D
Published: January 2020

The construction of a recombinant bacteriophage lambda library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pol gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the alpha and gamma herpesvirus families, lending further support to the notion that HHV-6 is a member of the beta herpesvirus group.
Host-derived 5' ends and overlapping complementary 3' ends of the two mRNAs transcribed from the ambisense S segment of Uukuniemi virus
Authors: Simons J F , Pettersson R F
Published: January 2020

Two mRNAs, coding for the N and NSS proteins, are transcribed from the small (S) Uukuniemi virus RNA segment by an ambisense strategy (J. F. Simons, U. Hellman, and R. F. Pettersson, J. Virol. 64:247-255, 1990). In this report, we describe the analysis of the 5' and 3' ends of the two mRNAs. Primer extension as well as cloning and sequencing of individual mRNAs showed that the 5' ends of both mRNAs contained nonviral sequences ranging from 7 to 25 residues in length (mean, 12 residues), indicating a cap-snatching mechanism similar to the one originally described for priming of influenza virus mRNA synthesis. In 35% of the cases, the first virion-specified nucleotide (an A residue) was substituted with a G residue. Between the translation termination codons of N and NSS, there is a 74-residue-long noncoding intergenic region (Simons et al., J. Virol. 64:247-255, 1990). Nuclease protection assays using both RNA and DNA hybridization probes showed that the 3' ends of the N and NSS mRNAs overlap each other by about 100 nucleotides. The 3' end of the NSS mRNA extends into the coding sequence of the N mRNA, whereas the N mRNA is terminated just prior to the stop codon of NSS. To our knowledge, this is the first example of overlapping complementary mRNAs in viruses with an ambisense coding strategy. No obvious transcription termination sequence was identified. However, because of a short palindromic sequence in the intergenic region, the 3' ends of both mRNAs (and consequently also the template RNAs) can be folded into an A/U-rich hairpin structure. It remains to be determined whether this structure plays any role in transcription termination.
Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies
Authors: Xu J Y , Gorny M K , Palker T , Karwowska S , Zolla-Pazner S
Published: January 2020

Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I.
Respiratory syncytial virus infection in anti-mu-treated mice
Authors: Graham B S , Bunton L A , Rowland J , Wright P F , Karzon D T
Published: January 2020

BALB/c mice were depleted of B cells by anti-mu treatment to investigate the pathogenesis of respiratory syncytial virus (RSV) infection in the absence of antibody. Termination of RSV replication after primary infection occurred with the same kinetics in anti-mu-treated mice as in phosphate-buffered saline (PBS)-treated controls. Yet, when rechallenged, anti-mu-treated mice were more permissive to RSV replication than PBS-treated controls. Anti-mu-treated mice also experienced greater illness than PBS-treated controls during both primary infection and rechallenge. Passive transfer of RSV-specific immune serum to anti-mu-treated mice before rechallenge reconstituted complete protection from RSV replication and diminished illness. Thus, RSV-specific antibody is not required to terminate RSV replication in primary infection, but without antibody, only partial immunity against rechallenge is induced. While it is unknown whether the mechanism is a direct effect on RSV titer or modulation of the illness-causing cellular immune response, the presence of RSV-specific antibody reduces illness in both primary RSV infection and rechallenge of mice.
Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system
Authors: Forger J M , Bronson R T , Huang A S , Reiss C S
Published: January 2020

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.
Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen
Authors: Weiner B M , Bradley M K
Published: January 2020

In an attempt to distinguish simian virus 40 (SV40) large T antigen (T) binding to ATP from hydrolysis, specific mutations were made in the ATP-binding site of T according to our model for the site (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Two acidic residues predicted to make contact with the magnesium phosphate were changed to alanines. The mutated T gene was completely defective for viral DNA synthesis and for virion production, and it was dominant defective for viral DNA replication. The defective T gene encoded a stable product (2905T) that oncogenically transformed mouse cell lines. 2905T, immunoprecipitated from transformed-cell extracts, bound SV40 origin DNA specifically and, surprisingly, it was active as an ATPase. A recombinant baculovirus was constructed for the production and purification of the mutant protein for detailed biochemical analyses. 2905T had only 10% of the ATPase and helicase of wild-type T. The Km of 2905T for ATP in ATPase assays was the same as the Km of wild-type T. ATP activated the ATPase activity of wild-type T, but not of 2905T. As tested by gel bandshift assay, 2905T bound to SV40 origin DNA and to individual sites I and II with affinities similar to that of the wild type. However, ATP did not modulate the DNA-binding activity of mutant T to site II. Therefore, this mutation in the ATP-binding site in T resulted in defects in the interaction between the protein and ATP that appeared to be responsible for the determination of the active state of T for DNA binding versus ATPase.
Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1
Authors: Montross L , Watkins S , Moreland R B , Mamon H , Caspar D L , Garcea R L
Published: January 2020

Polyomavirus normally assembles in the nucleus of infected mouse cells. Sf9 insect cells expressing the polyomavirus major capsid protein VP1 were examined by electron microscopy. Capsidlike particles of apparently uniform size were found in the nucleus. Immunogold electron microscopy demonstrated abundant VP1 in the cytoplasm which was not assembled into any recognizable higher-order structure. Cytoplasmic VP1 assembled after the cells were treated with the calcium ionophore ionomycin. Purified VP1 aggregates were shown by negative staining and cryoelectron microscopy to consist predominantly of particles similar to the empty T = 7 viral capsid. Thus, polyomavirus VP1 can assemble in vivo into capsids independent of other viral proteins or DNA. Nuclear assembly may result from increased available calcium in this subcellular compartment.
Isolation, identification, and partial cDNA cloning of genomic RNA of jaagsiekte retrovirus, the etiological agent of sheep pulmonary adenomatosis
Authors: York D F , Vigne R , Verwoerd D W , Querat G
Published: January 2020

The genome of the jaagsiekte (JS) retrovirus (JSRV), the etiological agent of sheep pulmonary adenomatosis (jaagsiekte), has been identified, isolated, and partly cloned. The JSRV genome is ca. 8.7 kb long. cDNA of the genomic RNA was synthesized and cloned. A clone, JS 46.1, was isolated and characterized. It has an insert of 2.1 kb which hybridizes to the same 8.7-kb RNA in all the JSRV-infected sheep lung washes tested but does not hybridize to maedi-visna virus, a sheep lentivirus often found coinfecting JSRV-infected lungs. Comparison of the amino acid sequence encoded by JS 46.1 with those encoded by other retroviruses revealed that JSRV has homology to the type D and B oncoviruses and to human endogenous retrovirus.
Cytopathic variants of an attenuated isolate of human immunodeficiency virus type 2 exhibit increased affinity for CD4
Authors: Hoxie J A , Brass L F , Pletcher C H , Haggarty B S , Hahn B H
Published: January 2020

Naturally occurring isolates of human immunodeficiency virus (HIV) have been described which are deficient in their ability to fuse with and kill CD4+ target cells. Although the molecular basis for their attenuation has not yet been defined, several lines of evidence point toward the viral envelope gene as a key determinant of viral pathogenicity. In the present article, we report the biological characterization of two highly cytopathic variants derived by repeated cell-free passage of an attenuated isolate of HIV type 2 (HIV-2), termed HIV-2/ST. Unlike the parental virus, the cytopathic variants were found to infect Sup-T1 cells with great efficiency and to induce both cell fusion and profound killing in these cultures. To determine whether changes in the viral envelope gene were responsible for the observed phenotypic differences, we examined the CD4 binding affinity of these viruses using a novel assay designed to quantitate the binding of fluoresceinated CD4 to viral envelope in its native configuration on the cell surface. The results demonstrated that the affinity of parental HIV-2/ST envelope for CD4 was 2 orders of magnitude reduced, while the cytopathic variants exhibited a high CD4 binding affinity, comparable to that of cytopathic HIV-1 and HIV-2 isolates. From these data, we conclude that the cytopathic potential of HIV depends, at least in part, on its receptor-binding affinity. In addition, our study documents strong selection pressures for viruses with increased CD4 affinity during propagation in immortalized T-cell lines, thus emphasizing the need to study HIV envelope biology in natural target cells.
A nested set of eight RNAs is formed in macrophages infected with lactate dehydrogenase-elevating virus
Authors: Kuo L L , Harty J T , Erickson L , Palmer G A , Plagemann P G
Published: January 2020

Total RNA was extracted from primary cultures of mouse macrophages isolated from 10-day-old mice 6 to 12 h postinfection with lactate dehydrogenase-elevating virus (LDV). Poly(A)+ RNA was extracted from spleens of 18-h LDV-infected mice. The RNAs were analyzed by Northern (RNA) blot hybridization with a number of LDV-specific cDNAs as probes. A cDNA representing the nucleocapsid protein (VP-1) gene located at the 3' terminus of the viral genome (E. K. Godeny, D. W. Speicher, and M. A. Brinton, Virology 177:768-771, 1990) hybridized to viral genomic RNA of about 13 kb plus seven subgenomic RNAs ranging in size from about 1 to about 3.6 kb. Two other cDNA clones hybridized only to the four or five largest subgenomic RNAs, respectively. In contrast, two cDNAs encoding continuous open reading frames with replicase and zinc finger motifs hybridized only to the genomic RNA. The replicase motif exhibited 75% amino acid identity to that of the 1b protein of equine arteritis virus (EAV) and 44% amino acid identity to those of the 1b proteins of coronaviruses and Berne virus. Combined, the results indicate that LDV replication involves formation of a 3'-coterminal-nested set of mRNAs as observed for coronaviruses and toroviruses as well as for EAV, with which LDV shares many other properties. Overall, LDV, like EAV, possesses a genome organization resembling that of the coronaviruses and toroviruses. However, EAV and LDV differ from the latter in the size of their genomes, virion size and structure, nature of the structural proteins, and symmetry of the nucleocapsids.
In vitro selection of variants of human immunodeficiency virus type 1 resistant to 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine
Authors: Gao Q , Gu Z X , Parniak M A , Li X G , Wainberg M A
Published: January 2020

Drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) have been isolated by in vitro selection. MT-4 cells were infected with either a laboratory strain (HIV-IIIB) or a clinical isolate (no. 187) of HIV-1 and maintained in medium containing subeffective concentrations of the drugs 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI). By gradually increasing the drug concentration in the culture medium during propagation of the virus on fresh MT-4 cells, we were able to isolate variants of HIV-IIIB and clinical isolate 187 which showed up to 100-fold increases in resistance to the drugs. The drug resistance phenotypes remained stable after propagation of the variants in the absence of drug pressure for over 2 months. However, variants resistant to one drug showed little or no cross-resistance to the other, suggesting that the genetic bases for resistance to the compounds differed. Genotypic analysis of these nucleoside-resistant variants by polymerase chain reaction (PCR) with primer pairs previously shown to correspond to mutations responsible for resistance to AZT was also carried out. A heterogeneity of genotypes was observed, with known mutations at pol codons 70 and 215 occurring in most of the AZT-resistant variants generated from either HIV-IIIB or clinical strain 187. However, mutations in codons 67 and 219 were less frequently detected, and none of these changes were observed in each of four variants resistant to ddI. Cloning and sequencing studies of the reverse transcriptase coding region of two of the isolates were also performed and confirmed the PCR data that had been obtained. In addition to previously described mutation sites responsible for resistance to AZT, an HIV-IIIB-resistant variant was shown to be mutated at positions 108 (Val----Ala) and 135 (Ile----Thr), while a resistant variant of strain 187 was mutated at positions 50 (Ile----Val) and 135 (Ile----Val).
Evidence for intracellular down-regulation of the epidermal growth factor (EGF) receptor during adenovirus infection by an EGF-independent mechanism
Authors: Hoffman P , Rajakumar P , Hoffman B , Heuertz R , Wold W S , Carlin C R
Published: January 2020

We have reported previously that human group C adenoviruses down-regulate the epidermal growth factor (EGF) receptor (EGF-R) (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). Expression of a 13.7-kDa protein encoded by a gene in the E3 transcription unit is necessary and sufficient for this effect (Carlin et al., Cell, 1989; B. L. Hoffman, A. Ullrich, W. S. M. Wold, and C. R. Carlin, Mol. Cell. Biol. 10:5521-5524, 1990). We show here that EGF-R down-regulation is accelerated in cells which overexpress the receptor when these cells are infected with virus mutants that overproduce the 13.7-kDa protein compared with wild-type virus. This is in contrast to EGF stimulation, for which others have shown that high concentrations of ligand are associated with low rates of receptor internalization in EGF-R-overexpressing cells (D. Kuppuswamy and L. J. Pike, J. Biol. Chem. 264:3357-3363, 1989; H. S. Wiley, J. Cell Biol. 107:801-810, 1988). We also show that the E3 protein is not present in media conditioned by infected cells and that it does not induce secretion of an EGF-like autocrine factor. Moreover, while mature membrane-bound EGF-R is down-regulated, the precursor of the membrane-bound form is not. Adenovirus infection also does not affect receptor-related molecules expressed in the secretory pathway. Interestingly, adenovirus-induced down-regulation is not regulated by concentrations of EGF associated with a slow rate of internalization in A431 cells. This suggests that 13.7-kDa protein expression triggers receptor entry by a novel ligand-independent pathway or, alternatively, that it compensates for a cellular factor that may be rate limiting during EGF-mediated endocytosis.
Transcriptional activation of several heterologous promoters by the E6 protein of human papillomavirus type 16
Authors: Desaintes C , Hallez S , Van Alphen P , Burny A
Published: January 2020

The E6 protein of human papillomavirus type 16 (HPV-16), along with E7, is responsible for the HPV-induced malignant transformation of the cervix. However, the mechanism of this transformation activity is not well understood. We investigated whether the entire E6 protein of HPV-16 could act as an activator of transcription. Experiments in which NIH 3T3 cells were cotransfected with an E6 expression vector together with the reporter chloramphenicol acetyltransferase (CAT) gene linked to various minimal promoters indicated that E6 could activate transcription from a series of viral TATA-containing promoters. Mutations or deletions that affected all upstream regulatory elements present in the thymidine kinase (TK) promoter, such as the GC and CAAT boxes, reduced the level of E6-induced transcription. However, compared with the basal level, these truncated promoters were still activated by E6. Although site-directed mutations of the TATA sequence present in the TK or human immunodeficiency virus long terminal repeat promoters reduced the level of basal transcription, they did not abolish the E6-mediated activation. Moreover, E6 could restore almost completely the full level of wild-type E6-induced transcription as long as the upstream regulatory elements (GC/CAAT in the TK promoter, NF-kappa B in the human immunodeficiency virus long terminal repeat) were intact. This dual interaction of HPV-16 E6 is reminiscent of the activity of a coactivator.
Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen
Authors: Deckhut A M , Lippolis J D , Tevethia S S
Published: January 2020

Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL.
Activation of the c-myb locus is insufficient for the rapid induction of disseminated avian B-cell lymphoma
Authors: Pizer E S , Baba T W , Humphries E H
Published: January 2020

We have previously reported that infection of 9- to 13-day-old chicken embryos with RAV-1 results in rapid development of a novel B-cell lymphoma in which proviral insertion has activated expression of the c-myb gene (E. Pizer and E. H. Humphries, J. Virol. 63:1630-1640, 1989). The biological properties of these B-cell lymphomas are distinct from those associated with the B-cell lymphomas that develop following avian leukosis virus proviral insertion within the c-myc locus. In an extension of this study, more than 200 chickens, infected as 10- to 11-day-old embryos, were examined for development of lymphomas that possess disrupted c-myb loci. Fourteen percent developed disseminated B-cell lymphoma. In the majority of these tumors, the RAV-1 provirus had inserted between the first and second exons that code for p75c-myb. However, insertions between the second and third exons and between the third and fourth exons were also detected. In situ analysis of myb protein expression in tumor tissue revealed morphological features suggesting that the tumor originates in the bursa. Within the bursa, the lymphoma appeared to spread from follicle to follicle without compromising the structural integrity of the organ. Tumor masses in liver demonstrated heterogeneous levels of myb protein suggestive of biologically distinct subpopulations. In contrast to the morbidity data, immunohistological analysis of bursae from 4- to 6-week-old chickens at risk of developing lymphomas bearing altered c-myb loci revealed lesions expressing elevated levels of myb in 16 of 19 birds. The activated myb lymphoma displayed very poor capacity to proliferate outside its original host. Only 1 of 33 in vivo transfers of tumor to recipient hosts established a transplantable tumor. None of the primary tumor tissue nor the transplantable tumor exhibited the capacity for in vitro proliferation. Similar experimental manipulation has yielded in vitro lines established from avian B-cell lymphomas expressing elevated levels of c-myc or v-rel. The dependence on embryonic infection for development of activated-myb lymphoma suggests a requirement for a specific target cell in which c-myb is activated by proviral insertion. It is likely, moreover, that continued tumor development requires elevated expression of myb proteins within a specific cell population in a restricted stage of differentiation.
Inhibition of human immunodeficiency virus type 1 Rev-Rev-response element complex formation by complementary oligonucleotides
Authors: Chin D J
Published: January 2020

Complementary 18-mer oligodeoxynucleotides (oligonucleotides) specifically inhibited the formation of human immunodeficiency virus Rev-Rev-response element (RRE) complexes. Inhibition of Rev-RRE binding required blockage of G-7819 to G-7820 in band shift assays. Structural studies revealed both local and distal effects. RRE structure was also disrupted by oligonucleotides targeted to other minor stems, by altering RNA renaturation conditions, or by reducing Rev concentrations--indicating a dynamic RRE structure and involvement of a minor RRE stem in the maturation of initial Rev-RRE complexes. Thus, complementary oligonucleotides alter RRE structure and may prove useful for the design of therapeutic anti-RRE oligonucleotides.
A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6
Authors: Littaua R A , Oldstone M B , Takeda A , Ennis F A
Published: January 2020

A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli. The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides. The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced. Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone.
Methylation of discrete sites within the enhancer region regulates the activity of the Epstein-Barr virus BamHI W promoter in Burkitt lymphoma lines
Authors: Jansson A , Masucci M , Rymo L
Published: January 2020

Eight of the nine viral antigens known to be expressed in in vitro Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines are downregulated in EBV-carrying Burkitt lymphomas (BL). Only EBNA1 can be detected in BL biopsies and BL-derived cell lines that maintain the representative phenotype during culture in vitro (group I BL lines). This restricted pattern of viral gene expression is accompanied by extensive EBV DNA methylation and can be reversed by treatment with the demethylating agent 5-azacytidine. Transcription of the genes encoding the six transformation-associated EBNAs can be initiated from one of two promoters located in the BamHI C and W regions, respectively, of the virus genome. We show that discrete sites within the BamHI W enhancer region are methylated in the group I BL lines Rael, Cheptage, and Elijah and become unmethylated after 5-azacytidine treatment that induces the expression of EBNA2. Demethylation correlates with activation of transcription from the BamHI W promoter as determined by S1 protection analysis. Reporter plasmids in which the W enhancer sequences were linked to the chloramphenicol acetyltransferase gene were active in untreated Rael, Cheptage, and Elijah cells, demonstrating that all of the required transcription factors are present in group I BL cells. Conversely, in vitro methylation of the enhancer sequences abolished their activity. The results suggest that methylation of control regions in the EBV genome may play a critical role for the regulation of viral gene expression in tumor cells.
Mapping of homologous, amino-terminal neutralizing regions of human T-cell lymphotropic virus type I and II gp46 envelope glycoproteins
Authors: Palker T J , Riggs E R , Spragion D E , Muir A J , Scearce R M , Randall R R , McAdams M W , McKnight A , Clapham P R , Weiss R A
Published: January 2020

Twelve synthetic peptides containing hydrophilic amino acid sequences of human T-cell lymphotropic virus type I (HTLV-I) envelope glycoprotein were coupled to tetanus toxoid and used to raise epitope-specific antisera in goats and rabbits. Low neutralizing antibody titers (1:10 to 1:20) raised in rabbits to peptides SP-2 (envelope amino acids [aa] 86 to 107), SP-3 (aa 176 to 189), and SP-4A (aa 190 to 209) as well as to combined peptide SP-3/4A (aa 176 to 209) were detected in the vesicular stomatitis virus-HTLV-I pseudotype assay. Higher-titered neutralizing antibody responses to HTLV-I (1:10 to 1:640) were detected with pseudotype and syncytium inhibition assays in four goats immunized with a combined inoculum containing peptides SP-2, SP-3, and SP-4A linked to tetanus toxoid. These neutralizing anti-HTLV-I antibodies were type specific in that they did not inhibit HTLV-II syncytium formation. Neutralizing antibodies in sera from three goats could be absorbed with peptide SP-2 (aa 86 to 107) as well as truncated peptides containing envelope aa 90 to 98, but not with equimolar amounts of peptides lacking envelope aa 90 to 98. To map critical amino acids that contributed to HTLV-I neutralization within aa 88 to 98, peptides in which each amino acid was sequentially replaced by alanine were synthesized. The resulting 11 synthetic peptides with single alanine substitutions were then used to absorb three neutralizing goat antipeptide antisera. Both asparagines at positions 93 and 95 were required for adsorption of neutralizing anti-HTLV-I antibodies from all three sera. Peptide DP-90, containing the homologous region of HTLV-II envelope glycoprotein (aa 82 to 97), elicited antipeptide neutralizing antibodies to HTLV-II in goats that were type specific. In further adsorption experiments, it was determined that amino acid differences between homologous HTLV-I and HTLV-II envelope sequences at HTLV-I aa 95 (N to Q) and 97 (G to L) determined the type specificity of these neutralizing sites. Thus, the amino-terminal regions of HTLV-I and -II gp46 contain homologous, linear, neutralizing determinants that are type specific.
Solubilization and promoter analysis of RNA polymerase from rice stripe virus
Authors: Barbier P , Takahashi M , Nakamura I , Toriyama S , Ishihama A
Published: January 2020

The RNA-dependent RNA polymerase associated with rice stripe virus was dissociated from viral RNA (vRNA) by CsCl centrifugation. The solubilized RNA-free RNA polymerase transcribed a model RNA template 50 nucleotides in length carrying the 5'- and 3'-terminal conserved sequences of all four genome RNA segments. A 3'-terminal half molecule of the model template was also active as a template. Hence, we propose that the 3'-terminal conserved sequence serves as a promoter for the rice stripe virus-associated RNA polymerase. The solubilized enzyme, however, was unable to transcribe vRNA. The failure of the solubilized enzyme to transcribe vRNA is discussed in relation to the apparent loss of RNA polymerase activity after treatment of virions with high concentrations of salt.
Phenotype-associated env gene variation among eight related human immunodeficiency virus type 1 clones: evidence for in vivo recombination and determinants of cytotropism outside the V3 domain
Authors: Groenink M , Andeweg A C , Fouchier R A , Broersen S , van der Jagt R C , Schuitemaker H , de Goede R E , Bosch M L , Huisman H G , Tersmette M
Published: January 2020

The nucleotide sequences of the env genes of eight phenotypically heterogeneous human immunodeficiency virus type 1 (HIV-1) clones recovered from a single individual within a 3-week period were compared. In addition, the accessory gene sequences for four of these clones were obtained. Variation among most accessory genes was limited. In contrast, pronounced phenotype-associated sequence variation was observed in the env gene. At least three of these clones most likely resulted from genetic recombination events in vivo, indicating that this phenomenon may account for the emergence of proviruses with novel phenotypic properties. Within the env genes of the eight clones, four domains could be defined, the sequence of each of which clustered in two groups with high internal homology but 11 to 30% cluster variation. The extensive env gene variation among these eight clones could largely be explained by the unique manner in which the alleles of these four domains were combined in each clone. Experiments with chimeric proviruses demonstrated that the HIV-1 env gene determined the capacity to induce syncytia and tropism for T-cell lines. Amino acids previously shown to be involved in gp120-CD4 and gp120-gp41 interaction were completely conserved among these eight clones. The finding of identical V3 sequences in clones differing in tropism for primary monocytes and T-cell lines demonstrated the existence of determinants of tropism outside the env V3 region.
Concerted integration of viral DNA termini by purified avian myeloblastosis virus integrase
Authors: Fitzgerald M L , Vora A C , Zeh W G , Grandgenett D P
Published: January 2020

Concerted integration of retroviral DNA termini, which produces a characteristic duplication of sequences at the integration site and formation of the proviral state, is a necessary step of the retroviral life cycle. We investigated the pairwise integration reaction catalyzed by purified avian retrovirus integrase by measuring the response to solution parameters and how the sequences of the viral termini, which comprise the avian imperfect inverted repeat, affect the reaction. When we optimized the reaction, an efficiency was achieved which approached that measured in systems using cytoplasmic extracts from virus-infected cells. The response of purified avian integrase to solution parameters was similar to that of the integration activity derived from cellular extracts. For strand transfer, the U3 viral terminal sequences were preferred to those of the U5 termini, a result we previously showed for the trimming reaction. That the sequence preference was the same for trimming and strand transfer may be further evidence that only one catalytic site is used for both reactions. A significant number of integration sites were sequenced. Interesting trends were found for the fidelity of the host duplications to the avian 6-bp duplication size, the clustering of the integration sites in the nonessential region of the lambda host DNA, and the sequence characteristics of the duplication sites.
Interference is controlled by segment 2 and possibly by segment 8 of the nondefective interfering influenza virus variant A/FM/1/47-MA
Authors: Brown E G , Dimock C F , Hannah K
Published: January 2020

On mouse adaption of A/FM/1/47, a variant, A/FM/1/47-MA (FM-MA), that had acquired the properties of increased virulence and interference was produced. Coinfection of cells with FM-MA and prototype strains of influenza virus yielded > 100-fold more FM-MA virus than prototype virus, whereas coinfection with the same prototype strains and the parental A/FM/1/47 virus produced equivalent yields, indicating that FM-MA had acquired mutations that confer the property of interference during mouse adaption. FM-MA is a nondefective interfering virus that grows to a high titer in vivo and in vitro. It has previously been shown that segments 4, 7, and 8 and possibly segment 5 account for the increased virulence. In this study we show by genetic analysis of FM-MA x A/HK/1/68 reassortants that segment 2, coding for the polymerase-associated protein PB1, and possibly segment 8, encoding the NS1 and NS2 proteins, control the ability of FM-MA to interfere. Interference could not be overcome by increasing the titer of the coinfecting strain, but delaying FM-MA infection by 4 to 6 h did avoid interference. During interference of A/HK/1/68, protein synthesis was inhibited by less than 65% throughout coinfection. Given the kinetics of interference and the small perturbation in protein synthesis, interference appeared to occur at the level of late genome replication or virus assembly. Virulence and interference in FM-MA were not linked. An interfering avirulent FM-MA x A/HK/1/68 reassortant, E07, was capable of protecting mice against lethal pneumonia due to a virulent noninterfering reassortant, H04.