bluuebunny/crossref_metadata_2025 huggingface dataset:
High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells
Authors: Boulerice F , Bour S , Geleziunas R , Lvovich A , Wainberg M A
Published: January 2020

Limiting-dilution techniques were employed to derive single-cell clones from U-937 cells that had been chronically infected with human immunodeficiency virus type 1. All clones thus obtained were positive for the presence of viral antigens; however, not all of the clones produced infectious progeny virus, as detected by the presence of reverse transcriptase (RT) activity in culture fluids. Six of these clones were monitored over time to determine whether their phenotype of human immunodeficiency virus type 1 expression was stable. Three clones maintained production of RT activity at a high level and showed a very high percentage of cells positive for viral p24 antigen, as determined by indirect immunofluorescence. The other three clones showed variations in either their levels of RT activity or the number of cells positive for p24, after which they stabilized. Infectious virus could be recovered from only three clones, as assessed by coculture experiments with different cell types. Two other clones were shown to produce noninfectious viruses. Molecular analyses at the DNA, RNA, and protein levels showed extensive variations between the viral isolates recovered from each clone.
Intraluminal proteolytic activation plays an important role in replication of type 1 reovirus in the intestines of neonatal mice
Authors: Bass D M , Bodkin D , Dambrauskas R , Trier J S , Fields B N , Wolf J L
Published: January 2020

Oral inoculation of suckling mice with reovirus serotype 1 (strain Lang) results in the conversion of intact virions to intermediate subviral particles (ISVPs) in the intestinal lumen. Digestion of virus in vitro with chymotrypsin or trypsin reveals two distinct forms of ISVPs, while the predominant species of ISVPs found in the small intestinal lumen appears to be identical to the chymotrypsin product. The in vivo conversion of virions to ISVPs was blocked by pretreatment of mice with protease inhibitors, resulting in inefficient replication of reovirus in intestinal tissue. The early inhibition of viral replication in suckling mice pretreated with protease inhibitors was not observed when suckling mice were inoculated with ISVPs generated by in vitro digestion with either chymotrypsin or trypsin. However, replication was decreased during secondary rounds of replication in mice receiving repeated doses of protease inhibitors, suggesting that luminal proteolytic digestion is important in rendering progeny virions infectious in the gut.
Immunosuppressive function of hepatitis B antigens in vitro: role of endoribonuclease V as one potential trans inactivator for cytokines in macrophages and human hepatoma cells
Authors: Jochum C , Voth R , Rossol S , Meyer zum Büschenfelde K H , Hess G , Will H , Schröder H C , Steffen R , Müller W E
Published: January 2020

The mRNAs of transiently expressed cytokine genes contain AUUUA-rich sequences in the 3' untranslated regions. In order to examine whether the AU-specific endoribonuclease V (EC 3.1.27.8) described previously by us transinactivates those mRNA species, we introduced a 51-nucleotide ATTTA sequence from tumor necrosis factor into the 3' untranslated region of beta-globin gene. Transcripts of that construct, synthesized in vitro, were prone to endoribonuclease V digestion at those AU-rich sequences. Stimulation of human macrophages with lipopolysaccharide resulted in a shift of the association state of the enzyme from the nuclear matrix-associated to the free form. This shift was strongly prevented by the hepatitis B surface antigen (HBsAg) and more weakly by hepatitis B nucleocapsid antigen and hepatitis B antigen of the X region. HBsAg and, to a lesser extent, hepatitis B nucleocapsid antigen and hepatitis B antigen of the X region inhibited the release of alpha interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony stimulating factor, while it had no effect on interleukin-1 production from stimulated macrophages. Using the human hepatoma cell line PLC/PRF/5, we provide further experimental evidence that endoribonuclease V acts in trans as a posttranscriptional inactivator for nuclear matrix-associated cytokine transcripts. These results suggest that those cytokine transcripts which contain reiterated (overlapping) AUUUA sequences are degraded by nuclear matrix-associated endoribonuclease V. This degradation was comparably high in cells incubated with HBsAg or cells which produced this antigen.
Prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (Acinonyx jubatus)
Authors: Heeney J L , Evermann J F , McKeirnan A J , Marker-Kraus L , Roelke M E , Bush M , Wildt D E , Meltzer D G , Colly L , Lukas J
Published: January 2020

The extent and progression of exposure to feline infectious peritonitis (FIP) virus in the cheetah, Acinonyx jubatus, was monitored by a world-wide serological survey with indirect fluorescent antibody titers to coronavirus. The indirect fluorescent antibody assay was validated by Western blots, which showed that all indirect fluorescent antibody-positive cheetah sera detected both domestic cat and cheetah coronavirus structural proteins. There was a poor correlation between indirect fluorescent antibody results and the presence of coronaviruslike particles in cheetah feces, suggesting that electron microscopic detection of shed particles may not be an easily interpreted diagnostic parameter for FIP disease. Low, but verifiable (by Western blots [immunoblots]) antibody titers against coronavirus were detected in eight free-ranging cheetahs from east Africa as well as from captive cheetahs throughout the world. Of 20 North American cheetah facilities screened, 9 had cheetahs with measurable antibodies to feline coronavirus. Five facilities showed patterns of an ongoing epizootic. Retrospective FIP virus titers of an FIP outbreak in a cheetah-breeding facility in Oregon were monitored over a 5-year period and are interpreted here in terms of clinical disease progression. During that outbreak the morbidity was over 90% and the mortality was 60%, far greater than any previously reported epizootic of FIP in any cat species. Age of infection was a significant risk factor in this epizootic, with infants (less than 3 months old) displaying significantly higher risk for mortality than subadults or adults. Based upon these observations, empirical generalizations are drawn which address epidemiologic concerns for cheetahs in the context of this lethal infectious agent.
Role of primary and secondary maternal viremia in transplacental guinea pig cytomegalovirus transfer
Authors: Griffith B P , Chen M , Isom H C
Published: January 2020

The modulation of the outcome of intrauterine guinea pig cytomegalovirus (GPCMV) infection by maternal viremia was investigated in the guinea pig model. Virus assay and in situ hybridization were used to study GPCMV infection of maternal blood, placentas, and fetuses following inoculation of pregnant guinea pigs by the subcutaneous, intracardiac, or intranasal route. Animals were inoculated in early gestation and were evaluated every 7 to 10 days throughout pregnancy. Although placental and fetal infections occurred in all groups examined, transfer of GPCMV to placentas and fetuses was most efficient in mothers inoculated subcutaneously. Primary viremia was followed by virus clearance from blood and by an episode of secondary viremia in the three groups of mothers examined. Placental and fetal infections in animals infected subcutaneously or intracardially were first detected at the time of primary viremia, persisted throughout gestation, and increased during secondary viremia. In contrast, placental and fetal infections in animals inoculated intranasally were demonstrated primarily during secondary viremia. Fetal infection was detected in all mothers with detectable primary and secondary viremia but in only 33% of mothers that experienced only primary viremia. These results suggest that secondary maternal viremia is associated with increased placental and fetal GPCMV infections.
Recombinant virus vaccine for bluetongue disease in sheep
Authors: Roy P , Urakawa T , Van Dijk A A , Erasmus B J
Published: January 2020

Bluetongue virus proteins derived from baculovirus expression vectors have been administered in different combinations to sheep, a vertebrate host susceptible to bluetongue virus, and the neutralizing antibody responses were measured. Vaccinated sheep were subsequently challenged, and the indices of clinical reaction were calculated. The results indicated that the outer capsid protein VP2 alone in doses of greater than 50 micrograms per sheep elicited protection. A dose of ca. 50 micrograms of VP2 protected some but not all sheep. However, when used in combination with ca. 20 micrograms of the other outer capsid protein, VP5, 50-micrograms quantities of VP2 not only protected all the vaccinated sheep but also elicited a higher neutralizing-antibody response. The addition of viral core proteins VP1, VP3, VP6, and VP7, the nonstructural proteins NS1, NS2, and NS3, and the outer capsid proteins VP2 and VP5 did not enhance this neutralizing-antibody response.
Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen
Authors: Wun-Kim K , Simmons D T
Published: January 2020

We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated. A 66-kDa fragment (131 to about 616) retained some activity, whereas a slightly smaller 62-kDa fragment (137 or 155 to 616) had none. This suggests that the minimal helicase domain maps from residue 131 to approximately residue 616. To map the helicase substrate-binding domain, we tested various fragments in a substrate-binding assay. The smallest fragment for which we could clearly demonstrate activity was a 46-kDa fragment (131 to 517). To determine the relationship between the helicase substrate domain and the origin-binding domain (131 to 257, minimal core region; 131 to 371, optimal region), we performed binding experiments with competitor DNAs present. We found that origin-containing double-stranded DNA was an excellent competitor of the binding of the helicase substrate to T antigen, suggesting that the two domains overlap. Therefore, full helicase activity requires at least a partial origin-binding domain as well as an active ATPase domain. Additionally, we found that the helicase substrate was a poor competitor of origin-binding activity, indicating that T antigen has a much higher affinity to origin sequences than to the helicase substrate.
Translational regulation of herpes simplex virus DNA polymerase
Authors: Yager D R , Marcy A I , Coen D M
Published: January 2020

Using as antigens fusion proteins expressed in bacteria, we have generated polyclonal antisera specific for the herpes simplex virus (HSV) type 1 DNA polymerase. A variety of immunologic, genetic, and biochemical assays were used to characterize these antisera and demonstrate their specificity for the HSV DNA polymerase. Using these antisera, measurements of the synthesis and accumulation of HSV DNA polymerase in infected Vero cells were made. Peak rates of polymerase synthesis were observed at 4 h postinfection, as much as 2 h before peak levels of polymerase mRNA accumulation. At all times examined, the HSV DNA polymerase polypeptide was found to be synthesized at a lower rate per mRNA than the viral thymidine kinase, with this difference being especially dramatic at later times. Infected-cell RNA isolated at 2 and 6 h postinfection directed the synthesis of similar amounts of polymerase polypeptide per polymerase transcript in rabbit reticulocyte lysates, indicating that polymerase transcripts are inherently as translatable at both times. An HSV mutant in which sequences including a short upstream open reading frame in the HSV DNA polymerase transcript were deleted specified polymerase mRNA whose translational efficiency was no more than marginally greater than that of the wild-type virus. These results demonstrate that polymerase expression is regulated by inefficient translation mediated by sequences other than the short upstream open reading frame and that this leads to an early shutoff of polymerase synthesis during HSV infection.
Inactivated simian immunodeficiency virus vaccine failed to protect rhesus macaques from intravenous or genital mucosal infection but delayed disease in intravenously exposed animals
Authors: Sutjipto S , Pedersen N C , Miller C J , Gardner M B , Hanson C V , Gettie A , Jennings M , Higgins J , Marx P A
Published: January 2020

Eight rhesus macaques were immunized four times over a period of 8 months with a psoralen-UV-light-inactivated whole simian immunodeficiency virus vaccine adjuvanted with threonyl muramyl dipeptide. Eight unvaccinated control animals received adjuvant alone. Only the vaccinated animals made antibodies before challenge exposure to the viral core and envelope as determined by Western blotting (immunoblotting) and virus-neutralizing antibodies. Ten days after the final immunization, one-half of the vaccinated and nonvaccinated monkeys were challenged exposed intravenously (i.v.) and one-half were challenge exposed via the genital mucosa with virulent simian immunodeficiency virus. All of the nonvaccinated control monkeys became persistently infected. In spite of preexisting neutralizing antibodies and an anamnestic antibody response, all of the immunized monkeys also became persistently infected. However, there was evidence that the clinical course in immunized i.v. infected animals was delayed. All four mock-vaccinated i.v. challenge-exposed animals died with disease from 3 to 9 months postchallenge. In contrast, only one of four vaccinated i.v. challenge-exposed monkeys had died by 11 months postchallenge.
Preferred crossover sites on polyomavirus DNA
Authors: Bourgaux P , Gendron D , Bourgaux-Ramoisy D
Published: January 2020

RmI is a hybrid replicon consisting of polyomavirus (Py) and mouse sequences that yields unit-length polyomavirus DNA via recombination between two directly repeated viral sequences of 182 base pairs (S repeats). To define the contribution of the S repeats in this intramolecular recombination, we derived from RmI a series of replicons containing the original S repeats as well as additional direct viral repeats which were 1 to 2 kilobases in length (L repeats). After mouse 3T6 cells were transfected with these constructs, recombination products that displayed the physical properties of homologous recombinants were detected. The structures of these recombinants indicated that whereas repeat length influences the likelihood of recombination, crossover occurs preferentially near the S repeats, provided that one of them is proximal to the viral origin of replication. This finding suggests that recombination near the S repeats depends on a process initiated near the viral origin of replication.
Bidirectional transcription of the Epstein-Barr virus major internal repeat
Authors: Rogers R P , Speck S H
Published: January 2020

In latently infected cell lines, the Epstein-Barr virus BamHI W fragment (major internal repeat) is transcribed in a rightward direction to yield exons common to several alternatively spliced messages which encode the six known viral nuclear antigens. A substantial steady-state population of very large (up to 20-kilobase) rightward transcripts is nuclear, much of it being polyadenylated. We report a rise in the levels of rightward transcripts hybridizing to BamHI-W sequences upon phorbol ester treatment of the clone-13 Burkitt's lymphoma cell line. We also report large (up to 15-kilobase) leftward transcripts hybridizing to BamHI-W sequences which occurred late in the viral lytic cycle in B95-8 and clone-13 cells. These leftward transcripts may antagonize the expression of the viral nuclear antigen messages by the formation of RNA duplexes.
Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus
Authors: Earl P L , Hügin A W , Moss B
Published: January 2020

The in vivo role of the proposed poxvirus early transcription termination signal TTTTTNT was confirmed by analysis of the RNA species made by recombinant vaccinia viruses. Premature transcription termination occurred following each of two TTTTTNT sequences present naturally within the coding region of the human immunodeficiency virus type 1 envelope gene. Alteration of the TTTTTNT sequences, without changing the encoded amino acids, resulted in production of full-length early mRNAs, improved protein expression, and a more consistent immune response.
A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins
Authors: Benko D M , Schwartz S , Pavlakis G N , Felber B K
Published: January 2020

We have characterized a novel 28-kilodalton protein, p28tev, detected in human immunodeficiency virus type 1-infected cells. tev is recognized by both tat and rev monospecific antibodies. tev is initiated at the tat AUG and contains the first exon of tat at its amino terminus, a small portion of env in the middle, and the second exon of rev at its carboxy terminus. A cDNA clone producing tev was cloned and expressed in human cells. Sequence analysis revealed that the tev mRNA is generated by splicing to a novel exon located in the env region. This identifies a fourth class of multiply spliced human immunodeficiency virus mRNAs, produced in infected and transfected cells. tev is regulated during the virus life cycle similarly to the other regulatory proteins, tat, rev, and nef, and displays both tat and rev activities in functional assays. Since tev contains important functional domains of tat and rev and is produced very early after transfection, it may be an important regulator in the initial phase of virus expression. Another rev-related protein, p18(6)Drev, containing env and rev sequences, was characterized and was found not to have detectable rev activity.
CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro
Authors: Cao Y Z , Friedman-Kien A E , Huang Y X , Li X L , Mirabile M , Moudgil T , Zucker-Franklin D , Ho D D
Published: January 2020

Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism.
Activation of a beta-galactosidase recombinant provirus: application to titration of human immunodeficiency virus (HIV) and HIV-infected cells
Authors: Rocancourt D , Bonnerot C , Jouin H , Emerman M , Nicolas J F
Published: January 2020

A quantitative bioassay for human immunodeficiency viruses has been developed on the basis of the ability of the tat gene to transactivate the expression of an integrated beta-galactosidase gene in a HeLa-CD4+ cell line. Infection by a single virion of HIV-1 or HIV-2 corresponds to a unique blue syncytium or a cell cluster detected after fixation and addition of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (a beta-galactosidase substrate). The number of infected lymphoid cells in a culture (stimulated human peripheral blood lymphocytes and cell lines) can also be quantified by cell-to-cell transmission of HIV into the HeLa-CD4(+)-beta-galactosidase monolayer. Infections by simian immunodeficiency viruses are similarly detected. This assay has been used to determine the dose response of drugs, the half-life of HIV at 37 degrees C, and the appearance of infectious particles after virus infection.
A deletion in the Friend spleen focus-forming virus env gene is necessary for its product (gp55) to be leukemogenic
Authors: Watanabe N , Nishi M , Ikawa Y , Amanuma H
Published: January 2020

To determine the biological significance of the 585-base-pair deletion in the env gene of Friend spleen focus-forming virus (SFFV) encoding a leukemogenic glycoprotein (gp55), we examined the pathogenicity of a constructed mutant SFFV (SFFVDF). In the SFFVDF genome, the env deletion was filled in with the corresponding env sequence of Friend mink cell focus-forming virus, whereas the 6-base-pair duplication and the single base insertion near the 3' terminus of SFFV env remained intact. SFFVDF was nonpathogenic in adult mice. During passage of SFFVDF through newborn mice, we recovered various pathogenic variant SFFVs. Molecular analyses of variant SFFV genome DNAs revealed the presence of a distinct deletion in each env gene, which was similar but not identical to that in the wild-type SFFV env. Starting with the SFFVDF genome DNA, other mutant SFFV genome DNAs were constructed in which the sequence coding for the gp70/p15E proteolytic cleavage site present in the SFFVDF genome was modified by oligonucleotide-directed site-specific mutagenesis to prevent the cleavage. These mutant SFFVs were also nonpathogenic. These results indicate that for the pathogenic activity of gp55, a certain env deletion is necessary which causes production of a gp70-p15E fusion protein with an absence of at least the N-terminal one-third of the p15E-coding region.
Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera
Authors: Frech B , Zimber-Strobl U , Suentzenich K O , Pavlish O , Lenoir G M , Bornkamm G W , Mueller-Lantzsch N
Published: January 2020

The terminal proteins TP1 and TP2 are putative products of Epstein-Barr virus (EBV) genes expressed during the latent cycle of the virus. They are predicted to code for 53- and 40-kilodalton integral membrane proteins. We used the baculovirus Autographa californica nuclear polyhedrosis virus as an expression vector to produce TP1 in large amounts in insect cells. The DNA sequences used to express TP1 originated from a TP1 cDNA derived from an M-ABA/CBL1 cDNA library. Rabbit antisera raised against procaryotic TP1 fusion proteins recognized a monomer and a dimer of the recombinant TP1 protein in the infected insect cells. Immunofluorescence studies of living insect cells showed that the recombinant protein is located in the plasma membrane. The insect cells infected with the recombinant baculovirus producing TP1 provided a test system to screen human antisera for TP1 antibodies. A total of 168 human EBV-positive and EBV-negative antisera were studied. TP1 antibodies were detected only in sera from nasopharyngeal carcinoma patients (16 out of 42). Rabbit antiserum raised against the recombinant TP1 protein expressed in the baculovirus system specifically recognized a protein of about 54 kilodaltons in the lymphoblastoid cell lines M-ABA and M-ABA/CBL1 and in the Burkitt's lymphoma cell lines BL18 and BL72. This protein could be located in the total membrane fraction of M-ABA cells and is upregulated by treating the cells with 12-O-tetradecanoylphorbol-13-acetate.
Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology
Authors: Uttenthal A , Larsen S , Lund E , Bloom M E , Storgård T , Alexandersen S
Published: January 2020

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.
Mapping of B-cell epitopes of the human hepatitis B virus X protein
Authors: Stemler M , Weimer T , Tu Z X , Wan D F , Levrero M , Jung C , Pape G R , Will H
Published: January 2020

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.
Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiency virus type 1
Authors: Fenouillet E , Gluckman J C , Bahraoui E
Published: January 2020

We have shown that enzymatic removal of N-linked glycans from human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoproteins gp160 and gp120 produced in BHK-21 cells did not significantly reduce their ability to bind to CD4, the cellular receptor for the virus. Because recombinant proteins may behave differently from proteins present on virions, we investigated whether such viral envelope glycoproteins either in a purified form or present on viral particles could be deglycosylated by treatment with an endoglycosidase F-N-glycanase mixture which cleaves all accessible glycan moieties. Endoglycosidase analysis of the carbohydrate composition of purified viral gp120 (vgp120) indicated a glycosylation pattern similar to that for recombinant gp120 (rgp120), and treatment with endoglycosidase F-N-glycanase resulted in comparable molecular weight (MW) reduction for both molecules. Similarly, after immunoblotting of the deglycosylated viral preparation, the characteristic 160- and 120-kilodalton (kDa) bands were replaced by 90- and 60-kDa bands, respectively. The apparent MW of gp41 shifted to 35 kDa. These results are consistent with complete deglycosylation. The immunoreactive conformation of envelope glycoproteins remained unaltered after deglycosylation: they were recognized to the same extent by specific human polyclonal or mouse monoclonal antibodies, and no proteolysis of viral proteins occurred during enzymatic treatment. Deglycosylation of vgp120 resulted in a less than 10-fold reduction of the ability to bind to CD4, presented either in a soluble form or at the cell membrane. In addition, deglycosylation significantly reduced, but did not abolish, HIV-1 binding to and infectivity of CD4+ cells as determined, respectively, by an indirect immunofluorescence assay and a quantitative dose-response infection assay. Taken together, these results indicate that removal of glycans present on mature envelope glycoproteins of HIV-1 diminishes but does not abolish either virus binding to CD4 or its capacity to infect CD4+ cells.
Identification of the Epstein-Barr virus terminal protein gene products in latently infected lymphocytes
Authors: Rowe D T , Hall L , Joab I , Laux G
Published: January 2020

The terminal protein (TP) gene produces two overlapping mRNAs in latently infected lymphocytes that are predicted to encode the similar polypeptides TP1 (497 amino acids) and TP2 (378 amino acids), with TP1 exon 1 providing 119 extra unique residues at the N terminus. Rabbit antisera were raised to procaryotic fusion proteins and used to detect expression of a predicted 53-kilodalton (kDa) TP product in transfected 293 cells and latently infected lymphocytes. Fractionation of transfected 293 cells showed this protein to be localized to an integral membrane preparation. The same fraction of latently infected lymphocytes contained proteins of 53 and 27 to 39 kDa as determined by Western immunoblotting with the TP-specific rabbit antisera. Immunoprecipitation of TP products from 35S-labeled human lymphoblastoid cells (CR/B95-8) was used in pulse-chase experiments and showed that TP1 was a labile protein with a half-life of approximately 2 to 4 h. The anti-fusion protein serum detected a 53-kDa TP1 and degradation products in the range of 25 to 35 kDa. A panel of Burkitt's lymphoma cell lines and cell lines established with virus recovered from the BL cells were analyzed by Western immunoblotting and found to contain the 53-kDa TP1 product, its degradation products, or both. Only two EBV-positive BL cell lines (BL72 and Wewak II) were negative in this assay. The results suggest that a labile TP1 protein may be expressed by most, if not all, EBV-infected cell lines.
Transcriptional activation of the adenine phosphoribosyltransferase promoter by an upstream butyrate-induced Moloney murine sarcoma virus enhancer-promoter element
Authors: Tang D C , Taylor M W
Published: January 2020

It has been documented that the activity of a specific promoter can be occluded by the presence of another promoter element upstream. We present evidence for a phenomenon contradictory to that predicted by the promoter occlusion theory. Transcription from the hamster aprt (adenine phosphoribosyltransferase) promoter was augmented instead of repressed in transfected mouse L cells when an upstream Moloney murine sarcoma virus enhancer-promoter element was induced with butyrate. Without an adjacent Moloney murine sarcoma virus element, butyrate could not activate the aprt promoter.
Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5' poly(A) leader on its early transcript
Authors: Ahn B Y , Jones E V , Moss B
Published: January 2020

The DNA-dependent RNA polymerase of vaccinia virus contains 8 to 10 virus-encoded polypeptides. We have mapped the gene encoding an 18-kilodalton RNA polymerase subunit to D7R, the seventh open reading frame of the HindIII D genomic subfragment. Localization of this gene was achieved by using antibody to the purified RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments. The identification was confirmed by translation of D7R transcripts made in vitro with bacteriophage T7 RNA polymerase. The phenotypes of two previously isolated conditionally lethal temperature-sensitive mutants that map to D7R (J. Seto, L. M. Celenza, R. C. Condit, and E. G. Niles, Virology 160:110-119, 1987) are consistent with an essential role of this subunit in late transcription. This polymerase gene, designated rpo18, predicts a polypeptide of 161 amino acids with a molecular mass of 17,892. The rpo18 gene is transcribed early in infection, even though the 5'-TAAATG-3' motif, which is conserved among many genes of the late class, is present near the RNA start site. Characterization of the 5' end of the early transcript by several different methods, including cDNA cloning, revealed a poly(A) leader with up to 14 adenylate residues, whereas only 3 are present in the corresponding location of the DNA template. Similar but somewhat longer poly(A) leaders have previously been observed in mRNAs of late genes. We noted a TAAATG motif near the initiation site of several other early genes, including the viral DNA polymerase, and carried out additional experiments to demonstrate that their early transcripts also have 5' poly(A) leaders. Thus, formation of the poly(A) leader is not exclusively a late function but apparently depends on sequences around the transcription initiation site.
Biological characterization of human monoclonal antibodies to rabies virus
Authors: Dietzschold B , Gore M , Casali P , Ueki Y , Rupprecht C E , Notkins A L , Koprowski H
Published: January 2020

Rabies virus antigen-specific human monoclonal antibodies (MAbs) that recognized either viral glycoprotein, ribonucleoprotein, or matrix proteins were generated. Only glycoprotein-specific MAb neutralized a variety of rabies viruses and protected laboratory rodents against lethal rabies virus infection. The determinant recognized by this MAb does not appear to reside in previously defined antigenic sites of the viral glycoprotein.
v-mos protein produced by in vitro translation has protein kinase activity
Authors: Herzog N K , Nash M , Ramagli L S , Arlinghaus R B
Published: January 2020

The v-mos protein, termed p37v-mos, has a closely associated serine/threonine protein kinase activity. To provide further information about its protein kinase activity, we tested the activity of p37v-mos produced in a cell-free translation system from transcripts generated from a cloned v-mos gene. Anti-mos(37-55) immunoprecipitates of in vitro-produced p37v-mos were found to possess serine/threonine protein kinase activity, whereas those obtained with anti-mos(260-271), known to block v-mos autophosphorylation, lacked kinase activity. The phosphorylated products were identical in size to p37v-mos and p43v-mos produced in protein kinase assays from Moloney murine sarcoma virus-infected cells expressing authentic p37v-mos. These results provide further proof that the protein kinase activity associated with p37v-mos is an intrinsic property of the v-mos gene product. This translation system also provides a useful experimental model to study the activation of the mos protein kinase. Thus, protein kinase assays performed on [35S]methionine-labeled p37v-mos produced p43v-mos at the expense of p37v-mos. Phosphatase treatment removed the p43v-mos species, resulting in increase of the p37v-mos-sized protein, confirming our previous interpretation that p43v-mos is a hyperphosphorylated form of p37v-mos.
Antigenic and genomic relationships among turkey and bovine enteric coronaviruses
Authors: Dea S , Verbeek A J , Tijssen P
Published: January 2020

Antigenic and genomic relationships among tissue culture-adapted turkey enteric coronavirus (TCV) isolates, three strains of avian infectious bronchitis virus (IBV), and mammalian coronaviruses were investigated. Immunoblotting and immunoprecipitation experiments using polyclonal antisera showed that the four major structural proteins of TCV cross-reacted with the four homologous proteins of bovine enteric coronavirus (BCV), the N and M proteins of mouse hepatitis virus serotype 3, and the N protein of IBV. Close antigenic relationships between TCV and BCV were also established by seroneutralization and hemagglutination-inhibition. Of 49 monoclonal antibodies produced against either TCV or BCV, 11 differentiated the two viruses. Five of these monoclonal antibodies had neutralizing activities and were directed to either the peplomeric S (gp200-gp100) or hemagglutinin HE (gp140-gp65) glycoproteins. BCV cDNA probes tested on purified viral preparations and coronavirus-positive (by electron microscopy) fecal samples from diarrheic turkey poults confirmed the relatedness of TCV and BCV. The two viruses produced distinct cytopathic changes in HRT-18 cells in the presence of trypsin, whereas only TCV isolates were able to reproduce the clinical symptoms in turkey poults. Their matrix (M) proteins undergo different glycosylation processes.
Isolation of a possible archetypal JC virus DNA sequence from nonimmunocompromised individuals
Authors: Yogo Y , Kitamura T , Sugimoto C , Ueki T , Aso Y , Hara K , Taguchi F
Published: January 2020

We molecularly cloned JC polyomavirus DNAs from urine samples of eight nonimmunosuppressed patients and two healthy individuals. The cloned viral DNAs all contained an archetypal regulatory sequence from which various regulatory sequences of JC polyomavirus isolates derived from patients with progressive multifocal leukoencephalopathy could have evolved by deletion and amplification.
Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates
Authors: Tomasselli A G , Hui J O , Sawyer T K , Staples D J , Bannow C A , Reardon I M , Chaudhary V K , Fryling C M , Pastan I , Fitzgerald D J
Published: January 2020

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.
Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein
Authors: Birnbaum F , Nassal M
Published: January 2020

As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.
Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein
Authors: Pavlovic J , Zürcher T , Haller O , Staeheli P
Published: January 2020

MxA and MxB are interferon-induced proteins of human cells and are related to the murine protein Mx1, which confers selective resistance to influenza virus. In contrast to the nuclear murine protein Mx1, MxA and MxB are located in the cytoplasm, and their role in the interferon-induced antiviral state was unknown. In this report we show that transfected cell lines expressing MxA acquired a high degree of resistance to influenza A virus. Surprisingly, MxA also conferred resistance to vesicular stomatitis virus. Expression of MxA in transfected 3T3 cells had no effect on the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1. Treatment of MxA-expressing cells with antibodies to mouse alpha-beta interferon did not abolish the resistance phenotype. The conclusion that resistance to influenza virus and vesicular stomatitis virus was due to the specific action of MxA is further supported by the observation that transfected 3T3 cell lines expressing the related MxB failed to acquire virus resistance.
The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression
Authors: Adami G R , Babiss L E
Published: January 2020

While the products of the type 5 adenovirus E1A and E1B genes can initiate pathways leading to a transformed rodent cell, little is known about how the rate of viral early gene expression influences the efficiency of this process. An adenovirus mutant [E1a(r) virus] that expresses its viral E1A and E1B genes at as much as a 100-fold-reduced rate relative to wild-type virus in infected CREF or HeLa cells transforms CREF cells at an 8-fold-higher efficiency than wild-type virus. Additional studies show that the reduction in viral E1A gene expression is solely responsible for this transformation phenotype, and at this low rate of viral E1A gene expression both E1A gene products must be expressed. Unlike previously characterized viruses which transform CREF cells at frequencies greater than wild-type virus, the foci obtained following E1a(r) virus infection were indistinguishable from those arising from wild-type virus by several criteria (morphological characteristics and anchorage-independent growth). Surprisingly, an analysis of viral early gene expression from a panel of wild-type- and E1a(r) virus-transformed CREF cell lines showed similar average rates of both viral E1A and E1B gene expression. By using an adenovirus-transformed cell line that is cold-sensitive for maintenance of the transformed cell phenotype, we show that both wild-type and the E1a(r) viruses can transform these cells at equally high efficiencies at the nonpermissive temperature of 32 degrees C. Our findings suggest that the process leading to a fully transformed cell involves multiple stages, with an early stage being facilitated by a reduced rate of viral E1A gene expression.
flvi-1, a common integration domain of feline leukemia virus in naturally occurring lymphomas of a particular type
Authors: Levesque K S , Bonham L , Levy L S
Published: January 2020

A locus in feline DNA, termed flvi-1, which may play an important role in the natural induction of lymphomas by feline leukemia virus (FeLV) was identified. Examination of a bank of 21 naturally occurring FeLV-positive feline lymphomas revealed that FeLV proviral integration occurs at flvi-1 in four independent tumors (19%). Independent integrations occurred within a 2.4-kilobase region of flvi-1, the probability of which by random chance can be estimated as 10(-16). Several lines of evidence, including sequence analysis of the long terminal repeat, demonstrated that proviruses integrated at flvi-1 are exogenously acquired and are oriented in the same transcriptional direction with respect to the locus. Molecularly cloned flvi-1 did not hybridize with probes representing several previously described proviral integration domains or with probes representing 10 oncogenes. The natural feline lymphomas examined in this study were heterogeneous with respect to tissue of origin, cell type, and number of monoclonal proviral integrations. The four tumors in which flvi-1 is interrupted were classified as members of a phenotypic subgroup containing seven lymphomas, i.e., at least four (57%) of seven lymphomas of this type contained FeLV proviral integration at flvi-1. Members of this phenotypic subgroup are non-T-cell lymphomas isolated from the spleen and contain an average of three proviruses, compared with an average of eight among all of the tumors examined. The small number of proviral integrations in tumors of this subgroup suggests that an early proviral integration event into flvi-1 can induce malignant change.
Localization of the membrane-associated region of vesicular stomatitis virus M protein at the N terminus, using the hydrophobic, photoreactive probe 125I-TID
Authors: Lenard J , Vanderoef R
Published: January 2020

The membrane-reactive, photoactivatable probe 125I-TID [3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-3H-diazirine] was found to label the M protein of vesicular stomatitis virus about 40% as much as G protein in intact virions, in agreement with labeling studies with other probes. By analyzing limited tryptic digestion and specific chemical cleavage products, the label was essentially entirely localized within the first 19, and probably within the first 5 to 10, amino acid residues at the N terminus, identifying this short amphipathic segment as the likely site of interaction of M protein with the viral bilayer.