bluuebunny/crossref_metadata_2025 huggingface dataset:
Viral protein R of human immunodeficiency virus types 1 and 2 is dispensable for replication and cytopathogenicity in lymphoid cells
Authors: Dedera D , Hu W , Vander Heyden N , Ratner L
Published: January 2020

Viral protein R (VPR) is conserved in human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). To assess its function, we have constructed mutations within the vpr coding regions of HIV-1 and HIV-2 predicted to express truncated VPR products. Infectious virus was produced by each proviral clone and showed similar replication kinetics and cytopathogenicity when compared with the corresponding parental proviral clone.
In vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein
Authors: Markoff L
Published: January 2020

Processing of dengue virus structural proteins was assessed in vitro. RNA transcripts for cell-free translation were prepared from cloned DNA (dengue virus type 4, strain 814669 genome) encoding capsid, pre-membrane (prM), and the first 23 amino acids of envelope (E). Processing of a 33-kilodalton precursor polypeptide encoded by wild-type RNA transcripts occurred only in the presence of added microsomal membranes. Under these conditions, cleavage at the capsid-prM and prM-E sites and glycosylation of prM occurred in association with translocation. Amino acid sequence analysis confirmed that translation initiated at the predicted N terminus of the capsid and that capsid-prM cleavage occurred at the predicted site for the action of signal peptidase following a candidate signal sequence (hydrophobic residues 100 to 113) in the dengue virus precursor. Mutations were introduced into the dengue virus DNA template by site-directed mutagenesis, altering nucleotide sequences encoding the capsid and the candidate signal for prM. The phenotypes of the mutants were deduced by analysis of the products of cell-free translation of the respective RNA transcripts. The resulting observations confirmed that cleavage at the capsid-prM and prM-E sites is effected entirely by signal peptidase and that the candidate signal is required for translocation.
Mapping of functional regions of murine retrovirus long terminal repeat enhancers: enhancer domains interact and are not independent in their contributions to enhancer activity
Authors: Hollon T , Yoshimura F K
Published: January 2020

We have used deletion and recombinant long terminal repeat (LTR) mutants to examine enhancer activity differences between LTRs of the nonpathogenic Akv and the thymus lymphomagenic MCF13 murine retroviruses. Deletion mutant analysis revealed that major control regions for MCF13 and Akv LTR enhancer activity were similar but not identical. For both LTRs, major control regions were distinctly different in a murine T-cell and a fibroblast cell line. Recombinant enhancer analysis showed that LTRs could be divided into three regions capable of altering the level of enhancer activity through cooperative or antagonistic interaction. The contribution of each region to enhancer activity was dependent on its context with respect to the other regions. LTR enhancer function in different cell types appears to be the result of the interaction of enhancer modular elements.
Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus
Authors: Nagesha H S , Brown L E , Holmes I H
Published: January 2020

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.
Use of TrpE/Gag fusion proteins to characterize immunoreactive domains on the human immunodeficiency virus type 1 core protein
Authors: Windheuser M G , Tegtmeier G E , Wood C
Published: January 2020

The human immunodeficiency virus (HIV) p24 core protein is one of the most immunogenic of HIV structural proteins. Infected individuals develop high titers of antibodies against p24 early in infection, which makes anti-p24 antibodies important serological markers. However, despite the clinical importance of the anti-p24 response, no systematic study to characterize the antigenic domains on the p24 protein has been reported. We report here on the use of 12 overlapping fragments of the HIV type 1 p24 protein, synthesized in bacteria as TrpE/Gag fusion proteins, to identify at least two and possibly three antigenic domains on the p24 protein. In addition, we note that different HIV-seropositive sera exhibited different patterns of reactivity with the p24 domains presented on our fusion proteins.
Identification and characterization of the woodchuck hepatitis virus origin of DNA replication
Authors: Seeger C , Maragos J
Published: January 2020

Replication of the woodchuck hepatitis virus (WHV) genome requires the synthesis of minus-strand DNA from an RNA template, the pregenome, by reverse transcription. During this reaction, the 5' end of minus-strand DNA becomes covalently linked to a protein. The position of the 5' end of minus-strand DNA was identified previously, but the initiation site for DNA synthesis on pregenomic RNA remained ambiguous because of a sequence repetition at the termini of the RNA template for reverse transcription. Employing a recently designed expression vector for the production of infectious WHV, we localized the origin of minus-strand DNA synthesis to the 3' end of pregenomic RNA. In addition, we identified the nucleotide sequences on pregenomic RNA that provide the signal for the initiation of reverse transcription. Removal of this signal sequence from pregenomic RNA abolished minus-strand DNA synthesis. Insertion of a DNA oligomer bearing this signal sequence at the 3' end of pregenomic RNA restored the production of minus-strand DNA joined to protein. Our results support a model in which protein is the primer for reverse transcription of minus-strand DNA of WHV.
The state of cellular differentiation determines the activity of the adenovirus E1A enhancer element: evidence for negative regulation of enhancer function
Authors: Herbst R S , Pelletier M , Boczko E M , Babiss L E
Published: January 2020

Most of the eucaryotic enhancer elements so far described consist of multiple DNA binding sites for proteins that act either synergistically or antagonistically to modulate the rate of transcription. In this report, we show that the activity of the adenovirus E1A enhancer element is suppressed in virus-infected undifferentiated rodent fetal fibroblast cells (CREF and F111 cells) and primary rat liver hepatocytes that have lost their fully differentiated phenotype (dedifferentiated). This contrasts with the results obtained for virus-infected differentiated or partially dedifferentiated rodent hepatocytes or hepatoma cell lines and human HeLa cells, in which deletion of the E1A enhancer domain greatly reduces the rate of E1A gene transcription. An in vitro quantitation of the nuclear proteins (from HeLa and CREF cells) that interact with and modulate the activity of the E1A enhancer revealed similar binding activities for the E2f and ATF proteins. However, an AP3-like (phi AP3) activity was present at a 10- to 20-fold higher concentration in CREF cells than in HeLa cells, and removal of this phi AP3-binding site on the viral genome resulted in an increase in the rate of E1A gene transcription in virus-infected CREF cells. Together, these results demonstrated that the factors which positively regulate enhancer function were present in CREF cells and that the phi AP3 factor was acting to suppress the activity of the E1A enhancer. Furthermore, the level of this factor was found to increase to even higher levels in CREF cells treated with 12-O-tetradecanoylphorbol-13-acetate, and this induction resulted in a further suppression in the rate of E1A gene transcription. On the basis of these observations, we propose that E1A expression is negatively regulated by the phi AP3 factor in undifferentiated rodent fetal fibroblast cells and that this could be an important mechanism that distinguishes between establishment of the differentiated cell versus transformed cell phenotypes.
Mutational analysis of human papillomavirus type 16 E7 functions
Authors: Watanabe S , Kanda T , Sato H , Furuno A , Yoshiike K
Published: January 2020

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.
Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus
Authors: Simons J F , Hellman U , Pettersson R F
Published: January 2020

We determined the complete nucleotide sequence of the small (S) RNA segment of Uukuniemi virus, the prototype of the Uukuvirus genus within the Bunyaviridae family. The RNA, which is 1,720 nucleotides long, contains two nonoverlapping open reading frames. The 5' end of one strand (complementary to the viral strand) encodes the nonstructural protein NSs (273 residues; molecular weight, 32,019), whereas the 5' end of the viral-sense strand encodes the nucleocapsid protein N (254 residues; molecular weight, 28,508). Thus, the S RNA uses an ambisense coding strategy previously described for the S segment of two phleboviruses and the arenaviruses. The localization of the N protein within the S RNA sequence was confirmed by amino-terminal sequence analysis of all five possible cyanogen bromide fragments obtained from purified N protein. Northern (RNA) blot analyses with strand-specific probes showed that the N and NSs proteins are translated from subgenomic mRNAs about 800 and 850 nucleotides long, respectively. These mRNAs are apparently transcribed from full-length S RNAs of opposite polarities. The two mRNA species were also detected in virus-infected cells. Interestingly, highly purified virions contained full-length S RNA copies of both polarities at a ratio of about 10:1. In contrast, virions contained exclusively negative-strand copies of the M RNA segment. The possible significance of these results for viral infection is discussed. The amino acid sequence of the N protein showed 35 and 32% homology (identity) with the N protein of Punta Toro and sandfly fever Sicilian viruses, two members of the Phlebovirus genus. The NSs proteins were much less related (about 15% identity). In addition, the extreme 5' and 3' ends of the S RNA, which are complementary to each other, also showed a high degree of conservation with the two phleboviruses. These results indicate that the uukuviruses and phleboviruses are evolutionarily related and suggest that the two genera could be merged into a single genus within the Bunyaviridae family.
Extramucosal spread and development of hepatitis in immunodeficient and normal mice infected with rhesus rotavirus
Authors: Uhnoo I , Riepenhoff-Talty M , Dharakul T , Chegas P , Fisher J E , Greenberg H B , Ogra P L
Published: January 2020

The pathogenic profiles of two heterologous animal rotaviruses, rhesus rotavirus strain MMU 18006 and bovine rotavirus strain WC3, were evaluated in mice with severe combined immunodeficiency (SCID mice) and normal BALB/c mice. Control animals were inoculated with homologous murine strain EDIM 5099 or a tissue culture-adapted murine rotavirus. Heterologous infection with rhesus rotavirus resulted in hepatitis in 84% of SCID and 21% of BALB/c mice, with mortality rates of 27 and 0%, respectively. Surviving SCID animals developed chronic liver disease, while symptoms in BALB/c mice resolved in 2 to 4 weeks after onset. Histopathologic examination revealed a diffuse hepatitis with focal areas of parenchymal necrosis. Rotavirus was detected in liver tissue from 100% of 29 SCID and 85% (11 of 13) BALB/c animals tested by cell culture infectivity, immunofluorescence, or electron microscopy. No extramucosal spread of virus or hepatitis was observed after infection with heterologous bovine strain WC3 or homologous murine rotaviruses. This finding of a novel rotavirus-induced disease manifestation suggests altered tissue tropism in a heterologous host for a group of viruses previously shown to replicate exclusively in the gut mucosa. The implications of our observations suggest that in human vaccine trials utilizing heterologous rotavirus strains, special attention should be paid to children with immunodeficiency disorders, and screening for hepatic function should be included in vaccine protocols.
Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains
Authors: Gorziglia M , Nishikawa K , Hoshino Y , Taniguchi K
Published: January 2020

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.
Translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' cap
Authors: Sedman S A , Gelembiuk G W , Mertz J E
Published: January 2020

The major late 16S mRNA species of simian virus 40 encodes both a 61-amino-acid protein, LP1, and the major virion protein, VP1. Although the initiation signal GCCAUGG is usually utilized at high efficiency, at least one-third of 40S ribosomal subunits bypass it when it is present on the major 16S mRNA of simian virus 40 (S. A. Sedman, P. J. Good, and J. E. Mertz, J. Virol. 63:3884-3893, 1989). The LP1 translation initiation codon is situated 10 bases from the 5' end of this mRNA. To determine whether the short length of the untranslated leader of this mRNA affects the efficiency of translation initiation at the LP1 initiation signal, monkey cells were transfected with plasmids which encode major late 16S-like mRNAs with 5' untranslated regions (UTRs) of 6 or 44 bases. Decreasing the length of the 5' UTR from 44 to 6 bases resulted in a 30% decrease in translation initiation at the LP1 AUG and a threefold increase in synthesis of VP1. When the VP1 open reading frame was replaced with the chloramphenicol acetyltransferase open reading frame, the reduction in 5' UTR length resulted in a 70% decrease in translation initiation at the LP1 AUG and a 30% increase in chloramphenicol acetyltransferase synthesis. Therefore, ribosomes bypass an AUG codon more efficiently when it is located very close to the 5' end of the mRNA.
Comparison of two host cell range variants of feline immunodeficiency virus
Authors: Phillips T R , Talbott R L , Lamont C , Muir S , Lovelace K , Elder J H
Published: January 2020

Two molecular clones of feline immunodeficiency virus were compared. The first clone, 34TF10, was from a Petaluma, Calif., isolate; the second, PPR, was isolated from a cat in the San Diego, Calif., area. The cats from which the isolates were obtained suffered from chronic debilitating illnesses. The two molecular clones differed in their in vitro host cell range. The 34TF10 clone infected the Crandall feline kidney and G355-5 cell lines, but replicated less efficiently on feline peripheral blood leukocytes. In contrast, the PPR clone productively infected the primary feline peripheral blood leukocytes but not Crandall feline kidney or G355-5 cells. The 34TF10 and PPR clones had an overall sequence identity of 91%. The env gene was the least conserved (85% at the amino acid level). Additionally, the potential open reading frame for a Tat-like protein, ORF 2, contained a stop codon in the 34TF10 isolate which was not found in the PPR clone. This truncation did not prevent in vitro or in vivo replication of 34TF10. Two splice acceptor sites were identified in the 34TF10 clone. One was 5' to the beginning of the putative tat open reading frame, and the other was 5' to the putative vif product. Both of these acceptor sites were conserved in the PPR clone. The long terminal repeats of the viruses were 7% divergent between the two clones, with a lack of conservation in putative NF-kappa B, LBP-1, and CCAAT enhancer-promoter sites.
Neutralization of poliovirus by cell receptors expressed in insect cells
Authors: Kaplan G , Freistadt M S , Racaniello V R
Published: January 2020

To examine the interaction of the poliovirus receptor (PVR) with virus and the role of the PVR in virus entry, the PVR was expressed in insect cells. Poliovirus bound to insect cells infected with a recombinant baculovirus (AcPVR) carrying cDNA encoding the PVR. Antibodies raised against PVR expressed in bacteria immunoprecipitated a 67-kilodalton polypeptide from cytoplasmic extracts of AcPVR-infected cells. Treatment of AcPVR-infected cells with tunicamycin revealed that the PVR is a glycoprotein containing N-glycosidic linkages and that carbohydrate accounts for nearly 50% of its molecular weight as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When PVR was solubilized from AcPVR-infected insect cells and incubated with poliovirus, viral infectivity was neutralized. Sedimentation analysis revealed that irreversibly altered 135S particles were formed after incubation of poliovirus at 37 degrees C with solubilized extracts of AcPVR-infected insect cells. These results demonstrate that poliovirus eclipse may result from interaction with the cell receptor at neutral pH in the absence of membranes and suggest that soluble receptors may be effective antiviral agents against picornaviruses.
Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting
Authors: Roth M J , Schwartzberg P , Tanese N , Goff S P
Published: January 2020

The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms. Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration.
Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen
Authors: Serunian L A , Auger K R , Roberts T M , Cantley L C
Published: January 2020

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.
Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor
Authors: Lacoste J , D'Addario M , Roulston A , Wainberg M A , Hiscott J
Published: January 2020

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.
Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells
Authors: Bosch F X , Schwarz E , Boukamp P , Fusenig N E , Bartsch D , zur Hausen H
Published: January 2020

The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells.
Regulated expression of nuclear genes by T3 RNA polymerase and lac repressor, using recombinant vaccinia virus vectors
Authors: Rodriguez D , Zhou Y W , Rodriguez J R , Durbin R K , Jimenez V , McAllister W T , Esteban M
Published: January 2020

Recombinant vaccinia viruses that express the bacteriophage T3 RNA polymerase (VV-T3pol) or the Escherichia coli lac repressor (VV-lacI) under control of the early-late vaccinia promoter P7.5 were constructed. To determine whether phage polymerase and lac repressor can function in the nucleus of mammalian cells, the bacterial chloramphenicol acetyltransferase (CAT) gene was cloned downstream of a T3 promoter (PT3-CAT) or downstream of a T3 promoter-lac operator fusion element (PT3Olac-CAT), and these reporter gene cassettes were introduced stably into NIH 3T3 or Ltk- cells. Infection of 3T3/PT3-CAT or Ltk-/PT3-CAT cells by VV-T3pol led to rapid expression of CAT (greater than 20 ng of CAT protein per 10(6) cells). The presence of hydroxyurea (which blocks virus DNA replication) did not prevent CAT production. When 3T3/PT3Olac-CAT cells were infected with both VV-T3pol and VV-lacI (multiplicities of infection of 2.5 and 10, respectively), greater than 30-fold repression of CAT gene activity by lac repressor was observed. This could be reversed to unrepressed levels by the presence of 10 mM o-nitrophenyl-beta-D-galactoside (IPTG) in the medium. Regulated expression of the target gene was observed with cell lines that had been maintained for over 1 year (greater than 50 passages in culture), and Southern blot analysis revealed the presence of the CAT gene only in the nuclear fraction in these cells, demonstrating the stability of the target gene. These results indicate that vaccinia virus-encoded proteins can function in the mammalian nucleus and provide the basis for a genetic system in which essential vaccinia virus genes, placed in the chromosome of a cell, can be used to complement defective virus particles. This approach may prove useful for other virus systems.
Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity
Authors: Simmons D T , Wun-Kim K , Young W
Published: January 2020

We have previously identified three regions (called elements) in the DNA-binding domain of simian virus 40 large tumor (T) antigen which are critical for binding of the protein to the recognition pentanucleotides GAGGC at the viral replication origin. These are elements A (residues 147 to 159), B1 (185 to 187), and B2 (203 to 207). In this study, we generated mutants of simian virus 40 in order to make single-point substitution mutations at nearly every site in these three elements. Each mutation was tested for its effect on virus replication, and T antigen was produced from all replication-negative mutants. The mutant proteins were assayed for binding to several different DNA substrates and for helicase activity. We found that within each element, mutations at some sites had major effects on DNA binding while mutations at other sites had moderate, mild, or minimal effects, suggesting that some residues are more important than others in mediating DNA binding. Furthermore, we provide evidence that certain residues in elements A and B2 (Ala-149, Phe-159, and His-203) participate in nonspecific double-stranded and helicase substrate (single-stranded) DNA binding while others (Ser-147, Ser-152, Asn-153, Thr-155, Arg-204, Val-205, and Ala-207) are involved in sequence-specific binding at the origin. The residues in element B1 (primarily Ser-185 and His-187) take part only in nonspecific DNA binding. The amino acids important for nonspecific DNA binding are also required for helicase activity, and we hypothesize that they make contact with the sugar-phosphate backbone of DNA. On the other hand, those involved in sequence-specific binding are not needed for helicase activity. Finally, our analysis showed that three residues (Asn-153 and Thr-155 in element A and Arg-204 in element B2) may be the most important for sequence-specific binding. They are likely to make direct or indirect contacts with the pentanucleotide sequences at the origin.
Transcriptional analysis of the eight-kilobase mRNA encoding the major capsid protein of human cytomegalovirus
Authors: Rudolph S A , Stamminger T , Jahn G
Published: January 2020

The 8-kilobase mRNA coding for the major capsid protein (MCP) of human cytomegalovirus was precisely mapped. Two 5' ends of the transcript were located within HindIII fragment a, 29 and 34 base pairs, respectively, downstream of the sequence TATTAGA. The 3' end was localized within HindIII fragment b of the viral genome. The MCP transcript was synthesized at late times after infection and was not detected before viral DNA replication. In addition, the MCP promoter region could be identified, which strongly responded to viral trans activation at early and late times after infection in a transient expression assay.
Effect of antiviral antibody on maintenance of long-term rubella virus persistent infection in Vero cells
Authors: Abernathy E S , Wang C Y , Frey T K
Published: January 2020

A Vero cell line with a long-term rubella virus persistent infection was maintained for 45 weeks in the presence of anti-rubella virus antibody of sufficient titer to completely neutralize the virus in the culture fluid to determine the effect of the presence of antibody on the maintenance of the persistent infection. Prior to antibody treatment, virus was continuously detected as plaque-forming units in the persistently infected culture fluid. Virus clones that were plaque purified from the persistently infected culture fluid were temperature sensitive and exhibited a reduced efficiency of replication and ability to induce cytopathic effects in Vero cells at the persistently infected culture temperature compared with the standard virus used to initiate the persistently infected culture. Defective interfering RNAs were the major intracellular virus-specific RNA species present in the persistently infected cells. Treatment with antibody failed to cure the persistently infected culture of virus, and the cells retained the ability to release virus after antibody treatment was discontinued. Interestingly, the presence of antibody led to the selection of a population of virus which was markedly less cytopathic for Vero cells than the virus population which was selected during persistent infection in the absence of antibody.
Thyroid hormone requirement for retinoic acid induction of mouse mammary tumor virus expression
Authors: Bolander F F , Blackstone M E
Published: January 2020

In normal mouse mammary epithelium, insulin, cortisol, and prolactin are absolute requirements for mouse mammary tumor virus expression. Retinoic acid further increased mouse mammary tumor virus expression two- to threefold but only when triiodothyronine was also present; neither retinoic acid nor triiodothyronine alone had any effect.
A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology
Authors: Clavel F , Orenstein J M
Published: January 2020

A deletion of 30 bases was created in the noncoding region of the human immunodeficiency virus type 1 genome that extends between the 5' splice donor and the start of the gag gene. Viral particles produced after transfection of this mutant provirus had a normal protein pattern but a reduced RNA content. The infectivity of the mutant virus was also markedly reduced but not completely abolished. Electron-microscopic examination of the mutant virions revealed major abnormalities of the nucleoid structure, mostly related to the dense material characteristic of mature particles, suggesting that the presence of RNA is essential to the normal structure of the nucleoid.
Molecular characterization of a rotaviruslike virus isolated from striped bass (Morone saxatilis)
Authors: Samal S K , Dopazo C P , McPhillips T H , Baya A , Mohanty S B , Hetrick F M
Published: January 2020

The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) were examined following purification of viruses from infected cell cultures. Virions had a double-layered capsid of icosahedral symmetry and a diameter of 75 nm. Purified viruses contained five polypeptides ranging in molecular mass from 130 to 35 kDa. None of the structural proteins were glycosylated. Treatment with EDTA did not remove the outer capsid. By using enzymes and a chaotropic agent, it was shown that VP5 was the most external polypeptide. The genome of SBR virus was composed of 11 segments of double-stranded RNA (dsRNA). The electrophoretic pattern of the dsRNA of SBR virus was different from that of reovirus type 1 (Lang) and rotavirus (SA11) dsRNA. The SBR virus was compared with reovirus type 1 and SA11 virus by RNA-RNA blot hybridization. There was no cross-hybridization between any of the genome segments of the SBR, reovirus type 1, or SA11 viruses. Antigenic comparison of SBR virus and SA11 virus by cross-immunoprecipitation and cross-immunofluorescence tests did not show any relationship. These results suggest that SBR virus could represent a new genus within the family Reoviridae.
The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation
Authors: Bartenschlager R , Junker-Niepmann M , Schaller H
Published: January 2020

Encapsidation of the pregenomic RNA into nucleocapsids is a selective process which depends on specific RNA-protein interactions. The signal involved in the packaging of the hepatitis B virus (HBV) RNA pregenome was recently defined as a short sequence located near the 5' end of that molecule (Junker-Niepmann et al., EMBO J., in press), but it remained an open question which viral proteins are required. Using a genetic approach, we analyzed whether proteins derived from the HBV P gene play an important role in pregenome encapsidation. The results obtained with point mutations, deletions, and insertions scattered throughout the P gene clearly demonstrate that (i) a P gene product containing all functional domains is required both for the encapsidation of HBV pregenomic RNA and for packaging of nonviral RNAs fused to the HBV encapsidation signal, (ii) known enzymatic activities are not involved in the packaging reaction, suggesting that P protein is required as a structural component, and (iii) P protein acts primarily in cis, i.e., pregenomic RNAs from which P protein is synthesized are preferentially encapsidated.
In vivo polyclonal B-lymphocyte activation elicited by murine viruses
Authors: Coutelier J P , Coulie P G , Wauters P , Heremans H , van der Logt J T
Published: January 2020

Viruses such as lactate dehydrogenase-elevating virus and adenovirus induce in vivo a polyclonal activation of murine B lymphocytes, followed by a marked increase in the production of immunoglobulin G2a (IgG2a). The role of T lymphocytes in this phenomenon was studied by injection of an anti-CD4 monoclonal antibody able to inhibit the T-helper function. This treatment profoundly depressed the production of IgG2a, whereas it had no effect on the proliferation of B cells. Activated B cells obtained from such infected and treated mice remained able to produce various immunoglobulin isotypes after exposure to an appropriate stimulus. In particular, gamma interferon, which is known to be secreted after viral infection, induced the production of IgG2a. These observations support the hypothesis that the influence of viruses on the switch of immunoglobulins is mediated by T-helper lymphocytes.
Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function
Authors: Long D , Cohen G H , Muggeridge M I , Eisenberg R J
Published: January 2020

We previously constructed seven mutations in the gene for glycoprotein D (gD) of herpes simplex virus type 1 in which the codon for one of the cysteine residues was replaced by a serine codon. Each of the mutant genes was cloned into a eucaryotic expression vector, and the proteins were transiently expressed in mammalian cells. We found that alteration of any of the first six cysteine residues had profound effects on protein conformation and oligosaccharide processing. In this report, we show that five of the mutant proteins exhibit temperature-sensitive differences in such properties as aggregation, antigenic conformation, oligosaccharide processing, and transport to the cell surface. Using a complementation assay, we have now assessed the ability of the mutant proteins to function in virus infection. This assay tests the ability of the mutant proteins expressed from transfected plasmids to rescue production of infectious virions of a gD-minus virus, F-gD beta, in Vero cells. Two mutant proteins, Cys-2 (Cys-106 to Ser) and Cys-4 (Cys-127 to Ser), were able to complement F-gD beta at 31.5 degrees C but not at 37 degrees C. The rescued viruses, designated F-gD beta(Cys-2) and F-gD beta(Cys-4), were neutralized as efficiently as wild-type virus by anti-gD monoclonal antibodies, indicating that gD was present in the virion envelope in a functional form. Both F-gD beta(Cys-2) and F-gD beta(Cys-4) functioned normally in a penetration assay. However, the infectivity of these viruses was markedly reduced compared with that of the wild type when they were preincubated at temperatures above 37 degrees C. The results suggest that mutations involving Cys-106 or Cys-127 in gD-1 confer a temperature-sensitive phenotype on herpes simplex virus. These and other properties of the cysteine-to-serine mutants allowed us to predict a disulfide bonding pattern for gD.
Factors affecting cellular tropism of human immunodeficiency virus
Authors: Kim S , Ikeuchi K , Groopman J , Baltimore D
Published: January 2020

To evaluate the basis of the slow growth of many human immunodeficiency virus strains in monocytes/macrophages, various stages of the virus life cycle have been studied for their possible contribution to viral tropism. Although we found that monocytic U937 cells had a higher percentage of CD4-positive cells than T-lymphoid H9 cells, the human immunodeficiency virus strain grew much less efficiently in the monocytic line. Viral tropism was primarily determined during the early stages of the virus cycle, that is, sometime between binding of the virus to the cell surface and reverse transcription of viral genomic RNA. Once the virus entered the host cell, reverse transcription, use of the long terminal repeat, RNA expression, and production of virus particles was about as efficient in monocytes as in T cells. Thus, during viral entry into the host cell cytoplasm there is a major limiting event that is particularly inefficient in U937 cells and possibly in all monocytes/macrophages.
Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites
Authors: Basu S , Varmus H E
Published: January 2020

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.
Association of simian virus 40 small-t antigen with the 61-kilodalton component of a cellular protein complex
Authors: Joshi B , Rundell K
Published: January 2020

Two cellular proteins, 61 and 37 kDa, are found in association with the simian virus 40 (SV40) small-t antigen. Fractionation in standard chromatography systems showed that these proteins were associated with one another in uninfected cells, suggesting that the small-t antigen may bind the complex as a whole and not each individual protein independently. In the presence of N-ethylmaleimide, the 37-kDA protein was selectively released from immune complexes, leaving the small-t antigen and 61-kDa protein in association. This result suggests that the small-t antigen may bind only the 61-kDa protein and that the 37-kDa protein may be associated with immune complexes by virtue of its association with the 61-kDa cellular protein.
Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome
Authors: Huang M , Jolicoeur P
Published: January 2020

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus.
Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome
Authors: Snyder R O , Im D S , Muzyczka N
Published: January 2020

We have demonstrated that when the covalently joined ends of linear adeno-associated virus (AAV) DNA are resolved in vitro, the virus-encoded Rep protein becomes covalently attached to the 5' ends of the DNA. The covalent bond is between a tyrosine residue of the AAV Rep protein and a 5' phosphate of a thymidine residue in the AAV genome. Only the Rep protein encoded by the AAV p5 promoter, Rep68, was capable of becoming covalently attached to the ends of the AAV genome; the Rep proteins encoded by the p19 promoter were not. We also investigated some of the requirements for the complete in vitro resolution reaction. Inhibitor studies suggested that terminal resolution required DNA polymerase delta, ATP, and the deoxyribonucleoside triphosphates but did not require the remaining ribonucleoside triphosphates, DNA polymerase alpha, RNA polymerase II, or topoisomerases I and II. Finally, purified AAV Rep68, when added to the crude cytosol from uninfected HeLa cells, was sufficient for resolution. This suggested that terminal resolution relies on host enzymes and the virus-encoded p5 Rep proteins.