bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody-mediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees
Authors: Emini E A , Nara P L , Schleif W A , Lewis J A , Davide J P , Lee D R , Kessler J , Conley S , Matsushita S , Putney S D
Published: January 2020

This study was undertaken to establish whether antibody directed against the human immunodeficiency virus type 1 (HIV-1) principal gp120 type-specific neutralization determinant can abolish the infectivity of HIV-1 in chimpanzees. Challenge inocula of the IIIb virus isolate were mixed in vitro with either immunoglobulin G (IgG) from an uninfected chimpanzee, nonneutralizing IgG from an HIV-seropositive human, a virus-neutralizing murine monoclonal antibody directed against the HIV-1 IIIb isolate, or virus-neutralizing IgG from a chimpanzee infected with the IIIb isolate. Both neutralizing antibodies were directed against the principal neutralization determinant of the challenge isolate. Establishment of infection following inoculation of each virus-antibody mixture into chimpanzees was assessed by virus-specific antibody development and by virus isolation. No protective effect was noted either with the control IgG or with the nonneutralizing anti-HIV IgG. By contrast, the polyclonal chimpanzee virus-neutralizing IgG prevented HIV-1 in vivo infection, while the neutralizing monoclonal antibody notably decreased the infectivity of the challenge virus. Hence, antibody to the gp120 principal neutralization determinant is able both to prevent HIV-1 infection in vitro and to inhibit infection in vivo.
Mutational analysis of the pseudoknot region in the 3' noncoding region of tobacco mosaic virus RNA
Authors: Takamatsu N , Watanabe Y , Meshi T , Okada Y
Published: January 2020

The approximately 200-nucleotide-long 3'-terminal noncoding region of tobacco mosaic virus (TMV) RNA contains a tRNA-like structure and, in its immediate upstream region, three consecutive pseudoknots, each of which is composed of two double-helical segments. To elucidate the biological functions of the pseudoknot region, we constructed several deletion mutant TMV-L (a tomato strain) RNAs by using an in vitro transcription system and tested their ability to multiply in both tobacco plants and protoplasts. When deletions were introduced just downstream of the termination codon of the coat protein gene in the 5'-to-3' direction progressively, five of six double-helical segments were dispensable for viral multiplication, indicating that the pseudoknot structures are not essential for multiplication. However, extension of the deletion into the central pseudoknot region resulted in reduction in viral multiplication, accompanied by loss of development of mosaic symptoms on systemic tobacco plants. Cessation of multiplication was observed when the sequence involved in formation of double-helical segment I just upstream of the tRNA-like structure was deleted irrespective of the start point and extent of deletion. Point mutations that destabilized double-helical segment I resulted in a loss or great reduction of viral multiplication, whereas the double mutants in which the double helix was restored by additional compensating base substitutions restored multiplication to nearly the wild-type level. Thus, double-helical segment I just upstream of the tRNA-like structure is a structural feature essential for viral multiplication.
Simian virus 40 DNA replication correlates with expression of a particular subclass of T antigen in a human glial cell line
Authors: Deminie C A , Norkin L C
Published: January 2020

Immunocytochemistry and in situ hybridization were used to identify simian virus 40 (SV40) large T-antigen expression and viral DNA replication in individual cells of infected semipermissive human cell lines. SV40 infection aborts before T-antigen expression in many cells of each of the human cell lines examined. In all but one of the human cell lines, most of the T-antigen-producing cells replicated viral DNA. However, in the A172 line of human glial cells only a small percentage of the T-antigen-expressing cells replicated viral DNA. Since different structural and functional classes of T antigen can be recognized with anti-T monoclonal antibodies, we examined infected A172 cells with a panel of 10 anti-T monoclonal antibodies to determine whether viral DNA replication might correlate with the expression of a particular epitope of T antigen. One anti-T monoclonal antibody, PAb 100, did specifically recognize that subset of A172 cells which replicated SV40 DNA. The percentage of PAb 100-reactive A172 cells was dramatically increased by the DNA synthesis inhibitors hydroxyurea and aphidicolin. Removal of the hydroxyurea was followed by an increase in the percentage of cells replicating viral DNA corresponding to the increased percentage reactive with PAb 100. The pattern of SV40 infection in A172 cells was not altered by infection with viable viral mutants containing lesions in the small t protein, the agnoprotein, or the enhancer region. Finally, in situ hybridization was used to show that the percentage of human cells expressing T antigen was similar to the percentage transcribing early SV40 mRNA. Thus, the block to T-antigen expression in human cells is at a stage prior to transcription of early SV40 mRNA.
Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus
Authors: Rice N R , Henderson L E , Sowder R C , Copeland T D , Oroszlan S , Edwards J F
Published: January 2020

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.
Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine
Authors: Dietzschold B , Gore M , Marchadier D , Niu H S , Bunschoten H M , Otvos L , Wunner W H , Ertl H C , Osterhaus A D , Koprowski H
Published: January 2020

We have mapped a linear epitope recognized by the virus-neutralizing monoclonal antibody 6-15C4 within the primary sequence of the G protein from the Evelyn-Rokitnicki-Abelseth strain of rabies virus. This was accomplished by using fragments of the rabies virus G protein and deduced amino acid sequences of neutralization-resistant variant rabies viruses. The monoclonal antibody 6-15C4 specifically recognized a synthetic peptide (peptide G5-24) which resembles the 6-15C4 epitope in structure. In addition, a tandem peptide constructed from the G5-24 peptide and a dominant TH cell epitope of the rabies virus N protein induced protective immunity against lethal rabies virus challenge infection in mice.
Herpesvirus saimiri U RNAs are expressed and assembled into ribonucleoprotein particles in the absence of other viral genes
Authors: Lee S I , Steitz J A
Published: January 2020

Marmoset T lymphocytes transformed by herpesvirus saimiri contain a set of five virally encoded U RNAs called HSUR1 through HSUR5. HSUR genes have been individually transfected into a nonlymphoid, nonsimian cell line (HeLa cells) in the absence of any other coding regions of the herpesvirus saimiri genome. The levels of HSUR1 through HSUR4 in HeLa transient-expression systems are comparable to those found in virally transformed T cells (23 to 91%). In contrast, HSUR5 is expressed at ninefold-higher levels in transfected HeLa cells. Immunoprecipitation experiments show that HSURs expressed in transfected cells bind proteins with Sm determinants and acquire a 5' trimethylguanosine cap structure, as they do in transformed T cells. HSUR1 or HSUR4 particles from transfected HeLa cells migrate between 10S and 15S in velocity gradients, identical to the sedimentation of "monoparticles" produced in virally transformed lymphocytes. We conclude from these transfection experiments that no other herpesvirus saimiri or host-cell-specific gene products appear to be required for efficient expression of the HSUR genes or for subsequent assembly of the viral U RNAs into small nuclear ribonucleoprotein particles. In lymphocytes transformed by herpesvirus saimiri, HSUR small nuclear ribonucleoprotein particles are involved in higher-order complexes that sediment between 20S and 25S. HSUR1, HSUR2, and HSUR5 dissociate from such complexes upon incubation at 30 degrees C, whereas the complex containing HSUR4 is stable to incubation.
Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7
Authors: Flores J , Sears J , Schael I P , White L , Garcia D , Lanata C , Kapikian A Z
Published: January 2020

We have synthesized 32P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.
Lack of myristoylation of poliovirus capsid polypeptide VP0 prevents the formation of virions or results in the assembly of noninfectious virus particles
Authors: Marc D , Masson G , Girard M , van der Werf S
Published: January 2020

We previously described the generation of a set of mutations into a cDNA of poliovirus type 1 in the myristoylation signal of the capsid polypeptide VP4 (D. Marc, G. Drugeon, A.-L. Haenni, M. Girard, and S. van der Werf, EMBO J. 8:2661, 1989). Genomic transcripts synthesized in vitro from the mutated cDNAs were found to be noninfectious upon transfection of permissive cells, and this property correlated with the lack of VP0 myristoylation in vivo. In the study presented here, we analyzed the assembly intermediates that could be recovered from cells transfected with the mutated transcripts. We found that 14S pentamers could still assemble to a certain extent with an unmyristoylated VP0. Furthermore, viral particles sedimenting at 150S and containing capsid polypeptides VP1 to VP4 and virus-specific RNA were detected in the transfected cells. However, these mature virions were less abundant than those recovered after transfection with an infectious transcript, and they were devoid of infectivity. The results suggest that VP0 myristoylation plays a role in the late steps of poliovirus assembly and that the myristate moiety of VP4 may be required in the early steps of poliovirus infection.
Sequence variability of bovine leukemia virus env gene and its relevance to the structure and antigenicity of the glycoproteins
Authors: Mamoun R Z , Morisson M , Rebeyrotte N , Busetta B , Couez D , Kettmann R , Hospital M , Guillemain B
Published: January 2020

The nucleotide sequences of the env genes of seven bovine leukemia viruses and the encoded peptide sequence were compared, with the objective of (i) determining the genetic distance separating bovine leukemia virus isolates from different geographical regions, (ii) identifying particular amino acids that contribute to the sequential and conformational epitopes, and (iii) relating such epitopes to their projected position in a three-dimensional model of the structure of the gp51 surface glycoprotein. Two bovine leukemia virus subgroups were clearly identified, a Japanese-American subgroup represented by strains lambda BLV-1, VdM, and FLK-BLV and a European subgroup by strains T15-2, LB285, and LB59. It was possible to identify amino acids that were important in determining three of the epitopes (F, G, and H) recognized by neutralizing monoclonal and polyclonal antibodies. On the model, these epitopes were adjacent and located on the exposed region of the molecule. Amino acid sequences contributing to a fourth cryptic epitope were identified; as predicted by the model, they lay on the opposite side to the neutralizable epitopes in a region involved in glycoprotein subunit association. The fact that this region is not normally exposed on the virion surface provides further evidence for the validity of the model.
Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis
Authors: Falgout B , Bray M , Schlesinger J J , Lai C J
Published: January 2020

The protective immunity conferred by a set of recombinant vaccinia viruses containing the entire coding sequence of dengue virus type 4 nonstructural glycoprotein NS1 plus various flanking sequences was evaluated by using a mouse encephalitis model. Mice immunized with recombinant vNS1-NS2a, which expresses authentic NS1, were solidly protected against intracerebral dengue virus challenge. However, mice immunized with recombinants vNS1-15%NS2a and vRSVG/NS1-15%NS2a, which express aberrant forms of NS1, were only partially protected (63 to 67% survival rate). Serologic analysis showed that mice immunized with vNS1-NS2a developed high titers of antibodies to NS1 as measured by radioimmunoprecipitation, enzyme-linked immunosorbent assay, and complement-mediated cytolytic assays. In addition, a pool of sera from these animals was protective in a passive transfer experiment. Lower titers of NS1-specific antibodies were detected in sera of animals immunized with vNS1-15%NS2a or vRSVG/NS1-15%NS2a by all three assays. These data support the view that protection against dengue virus infection in mice may be mediated at least in part by NS1-specific antibodies through a mechanism of complement-mediated lysis of infected cells. Additionally, immunization with two recombinant viruses expressing authentic NS1 of dengue virus type 2 conferred partial protection (30-50%) against dengue virus type 2 challenge.
Epstein-Barr virus BZLF1 trans activator induces the promoter of a cellular cognate gene, c-fos
Authors: Flemington E , Speck S H
Published: January 2020

The Epstein-Barr virus BZLF1 gene product ZEBRA is a DNA-binding protein that is partially homologous to c-Fos, binds specifically to AP-1 sites, and can induce the lytic cycle in latently infected B lymphocytes. Induction of the viral lytic cycle can also be achieved by treatment with the phorbol ester 12-O-tetrade-canoylphorbol-13-acetate, a reagent which activates gene expression in part through AP-1 (Jun/Fos). In this article the interrelationship between ZEBRA and AP-1 is extended by the demonstration that ZEBRA can induce c-Fos expression through AP-1 and "AP-1-like" sites present in the c-fos promoter. Induction of c-Fos may be necessary for the expression of other viral lytic genes and perhaps cellular genes whose products are required for viral replication.
Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus
Authors: Kniess N , Mach M , Fay J , Britt W J
Published: January 2020

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.
The three major immediate-early transcripts of bovine herpesvirus 1 arise from two divergent and spliced transcription units
Authors: Wirth U V , Vogt B , Schwyzer M
Published: January 2020

Among 54 transcripts expressed in a temporal cascade during lytic infection with bovine herpesvirus 1, we have previously identified three major immediate-early (IE) RNAs, IER4.2 (4.2 kb), IER2.9 (2.9 kb), and IER1.7 (1.6 to 1.8 kb depending on the virus strain) transcribed from the HindIII C genome region (U. V. Wirth, K. Gunkel, M. Engels, and M. Schwyzer, J. Virol. 63:4882-4889, 1989). Northern (RNA) blot, S1 nuclease protection, and primer extension analysis used in the present study demonstrated that all three IE transcripts were spliced and originated from two divergent transcription units with start sites located in the inverted repeat. Transcription unit 1 encoded two alternative spliced transcripts, IER4.2 and IER2.9, with a common exon 1 located at 0.797 to 0.795 map units (m.u.) and an exon 2 for IER4.2 (0.792 to 0.762 m.u.) in the inverted repeat; exon 2 for IER2.9 (0.754 to 0.738 m.u.) was located in the unique long sequence and transcribed in antisense orientation to latency-related RNA. Transcription unit 2 (0.818 to 0.836 m.u.), further characterized by cDNA cloning, encoded the spliced IER1.7 with three exons in the inverted repeat. Additional minor IE transcripts were interpreted as unspliced precursors and splicing variants. With regard to the number and layout of IE genes, bovine herpesvirus 1 occupies an intermediate position between pseudorabies virus and equine herpesvirus 1 on the one hand and varicella-zoster virus and herpes simplex virus type 1 on the other.
Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture
Authors: Schuitemaker H , Kootstra N A , de Goede R E , de Wolf F , Miedema F , Tersmette M
Published: January 2020

We previously demonstrated a correlation between the presence of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants showing tropism for cell line H9 and the occurrence of rapid CD4 cell decline and progression to AIDS. In contrast, in stable asymptomatic individuals, we detected only isolates with low replication rates that were non-syncytium-inducing (NSI) and nontropic for the H9 cell line. Here, we investigated the monocytotropism of established HIV-1 isolates with a panel of isolates and with biological HIV-1 clones with distinct phenotypes. Moreover, the prevalence and biological phenotypes of monocytotropic HIV-1 variants in the course of HIV-1 infection were analyzed in comparative primary isolation studies on peripheral blood lymphocytes (PBL) and monocyte-derived macrophages (MDM). In cell-free infection studies with MDM from eight blood donors, 13 of 17 NSI isolates but only 4 of 14 SI isolates were able to infect MDM. NSI isolates also infected significantly more different donors than SI variants (median, 3 of 8 versus 0 of 8). This enhanced monocytotropism of NSI isolates was confirmed in experiments with biological HIV-1 clones with distinct phenotypes recovered from the same donor. To investigate the prevalence and biological phenotypes of monocytotropic variants in different stages of HIV-1 infection, sequential isolates from peripheral blood mononuclear cell samples from nine asymptomatic individuals, five of whom progressed to AIDS and seven of whom had a known time of seroconversion, were recovered by cocultivation with both PBL and MDM. Monocytotropic variants were obtained from 37 of 42 time points. All monocytotropic variants were NSI in PBL culture and non-T-cell-line tropic, even when SI, T-cell-line-tropic HIV-1 variants could be recovered from the same patient sample by cocultivation with PBL. We conclude that monocytotropic HIV-1 variants mostly have an NSI phenotype in PBL and, in contrast to SI variants, are present at all stages of HIV-1 infection. These results suggest an important role for monocytotropic variants in the persistence of HIV-1 infection.
Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein
Authors: Ornelles D A , Shenk T
Published: January 2020

The distribution of the adenovirus early region 1B 55-kDa protein (E1B-55kDa) in lytically infected HeLa cells was determined. At the time of infection, when the E1B-55kDa protein facilitates the cytoplasmic accumulation of viral mRNA while simultaneously restricting the accumulation of most cellular mRNA, five distinct intracellular localizations of the protein were observed. Only one of these was disrupted when cells were infected with a mutant virus that fails to produce a second viral protein encoded by early region 4 (E4-34kDa). This protein normally forms a complex with the E1B-55kDa polypeptide, enabling it to influence RNA metabolism. This key localization of the E1B protein was within and about the periphery of nuclear viral inclusion bodies believed to be the site of viral DNA replication and transcription. In the absence of the E4-34kDa protein, the coincidence of E1B-55kDa-specific immunofluorescence and phase-dense viral inclusions was reduced compared with that in a wild-type infection. Similarly, by immunoelectron microscopy, the relative number of E1B-55kDa-specific immunogold particles associated with the clear fibrillar inclusion bodies was reduced. However, the E4-34kDa protein was not required for the close association of the early region 2A DNA binding protein with the viral inclusions. We propose that the viral 55-kDa-34-kDa protein complex interacts with a cellular factor required for cytoplasmic accumulation of mRNAs and directs it to the periphery of the transcriptionally active viral inclusion bodies. This model provides an explanation for the ability of these viral proteins to simultaneously enhance accumulation of viral mRNAs and inhibit accumulation of cellular mRNAs.
Complex formation between the lymphotropic papovavirus large tumor antigen and the tumor suppressor protein p53
Authors: Symonds H , Chen J D , Van Dyke T
Published: January 2020

The simian B-lymphotropic papovavirus (LPV) encodes a large tumor antigen (T antigen) which is 45% identical to both the simian virus 40 (SV40) and the polyomavirus (PyV) large T antigens. In transgenic mice, the transforming properties of the LPV T antigen are similar to those of the SV40 T antigen. However, little is known about its biochemical activities. Since SV40 T antigen forms a complex with and stabilizes the host cell tumor suppressor protein p53 while the PyV large T antigen does not, we characterized the LPV T antigen for its ability to complex p53. We demonstrate an association between LPV T antigen and p53 in both a tumor-derived cell line and BALB/c 3T3 cells transformed in culture. A third protein of approximately 68 kDa which was found associated with the LPV T antigen-p53 complex in tumor-derived cells appears to be heat shock protein 70 (hsp70). The half-life of p53 in all LPV T-antigen-transformed cells was extended significantly; i.e., it was 3 to 7 h compared with 19 minutes in BALB/c 3T3 cells. The half-life of the LPV T antigen itself was 5 to 9 h depending on the cell line origin. That p53 was stabilized because of association with LPV T antigen and not because of mutation was demonstrated with the p53 conformation-dependent monoclonal antibody PAb246. This antibody distinguishes between wild-type p53 (PAb246+) and mutant, oncogenic p53 (PAb246-). Sequential immunoprecipitation showed all detectable p53 to be of the PAb246+ class in each LPV-transformed cell line, suggesting that the stable p53 was indeed wild type.
Sequence-specific interaction of the Ets1 protein with the long terminal repeat of the human T-lymphotropic virus type I
Authors: Gitlin S D , Bosselut R , Gégonne A , Ghysdael J , Brady J N
Published: January 2020

We recently demonstrated that members of the c-ets proto-oncogene family, Ets1 and Ets2, are sequence-specific transcriptional activators of the human T-lymphotropic virus type I (HTLV-I) long terminal repeat (LTR). We now report that the HTLV-I LTR contains two distinct Ets1-responsive regions, ERR-1 and ERR-2. Expression of Ets1 with reporter plasmids containing ERR-1 or ERR-2 upstream of a basal promoter resulted in an increase in transcriptional activity. By gel mobility shift assay, the interaction of Ets1 with the downstream ERR-1-binding region was found to be more stable than its interaction with the upstream ERR-2 region. By DNase I footprint, gel mobility shift, and methylation interference analyses, ERR-1 was found to contain two Ets1 binding sites, ERE-A and ERE-B. A recombinant Ets1 protein was found to bind with higher affinity to ERE-A than to ERE-B. Binding of Ets1 to these sites appears to result in a specific and sequential protection of a 37-nucleotide sequence of the HTLV-I LTR from -154 to -118. In view of the high-level expression of Ets1 in lymphoid cells, the c-ets proto-oncogenes encode transcription factors which could play an important role in both basal and Tax1-mediated HTLV-I transcription.
Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme
Authors: Weerasinghe M , Liem S E , Asad S , Read S E , Joshi S
Published: January 2020

Toward gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections in AIDS, Moloney murine leukemia virus-derived retroviral vectors were engineered to allow constitutive and tat-inducible expression of an HIV-1 5' leader sequence-specific ribozyme (Rz1). These vectors were used to infect the human CD4+ lymphocyte-derived MT4 cell line. The stable MT4 transformants expressing an HIV-1 RNA-specific ribozyme, under the control of the herpes simplex virus thymidine kinase (tk) promoter, were found to be somewhat resistant to HIV-1 infection as virus production was delayed. In cells allowing ribozyme expression under control of the simian virus 40 or cytomegalovirus promoter, the rate of HIV-1 multiplication was slightly decreased, and virus production was delayed by about 14 days. The highest level of resistance to HIV-1 infection was observed in MT4 cells transformed with a vector containing a fusion tk-TAR (trans activation-responsive) promoter to allow ribozyme expression in a constitutive and tat-inducible manner; no HIV-1 production was observed 22 days after infection of these cells. These results indicate that retroviral vectors expressing HIV-1 RNA-specific ribozymes can be used to confer resistance to HIV-1 infection.
Mouse hepatitis virus S RNA sequence reveals that nonstructural proteins ns4 and ns5a are not essential for murine coronavirus replication
Authors: Yokomori K , Lai M M
Published: January 2020

Genes 4 and 5 of mouse hepatitis virus (MHV) are known to encode nonstructural proteins ns4, ns5a, and ns5b, whose function is unknown. In this study, we demonstrated that one of the MHV strains, MHV-S, did not synthesize mRNA 4 and made a smaller mRNA 5. Sequence analysis showed that the transcription initiation site for gene 4 of MHV-S was mutated from the consensus UCUAAAC to UUUAAAC, consistent with the idea that mutations in this region abolish mRNA synthesis. Furthermore, within gene 5 there were deletions totaling 307 nucleotides which deleted almost all of open reading frame 5a, but preserved open reading frame 5b of gene 5. Comparison of the growth of MHV-S with other MHV strains in DBT cells revealed no significant growth defect in MHV-S. These results suggest that ns4 and ns5a are not essential for viral replication in tissue culture cells, and thus join gene 2 and the hemagglutinin-esterase (HE) gene as nonessential viral genes in MHV.
Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation
Authors: Maldarelli F , Martin M A , Strebel K
Published: January 2020

cis-acting inhibitory region (IR) sequences were identified within the gag/pol gene of the human immunodeficiency virus type 1 (HIV-1) by using a novel feedback-stimulated, rev-independent tat reporter gene to screen HIV-1 sequences in transient expression assays. Two regions, a 1,295-nucleotide segment in the gag gene (IR-1) and a 1,932-nucleotide segment of the pol gene (IR-2), each inhibited reporter gene expression 10- to 20-fold. IR-1 and IR-2 both contained subsequences which inhibited reporter gene expression. Introduction of IR sequences into a heterologous reporter plasmid, pCMV-CAT, resulted in decreased chloramphenicol acetyltransferase expression, suggesting that the inhibitory effect was not restricted to a reporter gene under the control of the HIV-1 promoter. The presence of HIV IR sequences in cis did not alter relative levels of reporter gene RNA; however, fractionation studies revealed IR-containing RNA accumulated in the nucleus. These findings demonstrate that IR sequences within the gag/pol region affect gene expression by altering the cellular distribution of viral RNA.
Herpes simplex virus 1 RNA-binding protein US11 negatively regulates the accumulation of a truncated viral mRNA
Authors: Roller R J , Roizman B
Published: January 2020

The US11 gene of herpes simplex virus 1 (HSV-1) encodes a site-specific, basic, RNA-binding protein. The viral RNA sequences bound by US11 protein precipitated by a monoclonal antibody hybridized to a 1.3-kb BamHI C' fragment of the HSV-1 genome. This fragment encodes a US11-regulated transcript which accumulates to high level in the cells infected with US11- virus but not in cells infected with wild-type virus. This transcript, designated delta 34, is a truncated form of the mRNA encoding an essential protein encoded by the UL34 open reading frame. The US11 protein was shown to bind delta 34 RNA at or near its 3' terminus. The nucleotide sequence of the region surrounding the termination of transcription of delta 34 RNA transcription suggests that the latter may be the product of transcriptional attenuation. US11 protein resembles the tat protein of human immunodeficiency virus with respect to size, charge, nucleolar accumulation, and possibly effect on accumulation of its target RNA but does not share with it discernible sequence homology.
Demonstration of two distinct cytopathic effects with syncytium formation-defective human immunodeficiency virus type 1 mutants
Authors: Dedera D , Ratner L
Published: January 2020

The mechanism of human immunodeficiency virus type 1 (HIV-1) cytopathicity is poorly understood and might involve formation of multinucleated giant cells (syncytia), single-cell lysis, or both. In order to determine the contributions of the fusion domain to syncytium formation, single-cell lysis, and viral infectivity and to clarify the molecular details of these events, insertion mutations were made in the portion of env encoding this sequence in the functional HIV-1 proviral clone HXB2. Viruses produced from these mutant clones were found to have a partial (F3) or complete (F6) loss of syncytium-forming ability in acutely infected CEM, Sup T1, and MT4 T-cell lines. During the early stage of acute infection by F6 virus, there was a loss of the syncytial cytopathic effect, which resulted in increased cell viability, and a 1.9- to 2.6-fold increase in virus yield in the cell lines tested. In the late stage of acute infection, the single-cell cytopathic effect of F6 virus was similar to that of the parental HXB2 virus. The F3 and F6 viruses were also found to have a 1.7- to 43-fold reduction in infectivity compared with the HXB2 virus. The mutant F3 and F6 and parental HXB2 envelope proteins were expressed in vaccinia virus, and the mutant envelope proteins were observed to be defective in their ability to form syncytia. BSC-40 cells infected with vaccinia virus recombinants revealed no differences in kinetics of cleavage, cell surface expression, or CD4 binding capacity of the mutant and parental envelope proteins. These results demonstrate that a loss of syncytium formation results in an attenuation of infectivity and a loss of the syncytial cytopathic effect without a loss of single-cell lysis. These mutants may reflect in tissue culture the changes observed in the HIV isolates in vivo during disease progression, which exhibit marked differences in syncytium production.
Borna disease: association with a maturation defect in the cellular immune response
Authors: Carbone K M , Park S W , Rubin S A , Waltrip R W , Vogelsang G B
Published: January 2020

Borna disease virus (BDV) is a negative-strand RNA virus which produces persistent infection in a variety of experimental animals. In the rat, the presence or absence of clinical signs of Borna disease, a characteristic, biphasic neurobehavioral illness, depends on host-related factors. A window of opportunity exists after birth wherein inoculation with BDV produces a persistently infected rat without signs of Borna disease or encephalitis (persistent, tolerant infection-newborn [PTI-NB] rat). Although immunopathological destruction of the nervous system does not occur in the PTI-NB rat, significant alterations in the development of the nervous system were noted, including site-specific lysis of neurons. Unlike the case with other pharmacologically produced, persistent, tolerant BDV infections, adoptive transfer of spleen cells from BDV-infected rats did not produce disease in the PTI-NB rats. PTI-NB rats developed Borna disease after being connected by parabiosis to rats with Borna disease. Bone marrow transplantation experiments revealed that bone marrow cells from PTI-NB rats produced Borna disease in lethally irradiated, BDV-infected recipient rats. Bone marrow from PTI-NB rats contained a complement of inflammatory cells capable of inducing Borna disease. Thus, the loss of BDV-specific cellular immunity appeared to occur after the release of cells from the bone marrow.
Dissecting the activating mutations in v-erbB of avian erythroblastosis virus strain R
Authors: Shu H K , Pelley R J , Kung H J
Published: January 2020

The v-erbB oncogene isolated from the R (or ES4) strain of avian erythroblastosis virus is capable of inducing erythroleukemia and fibrosarcomas. This oncogene differs from the proto-oncogene c-erbB, the avian homolog of the epidermal growth factor receptor, by its lack of an intact ligand-binding domain as well as additional alterations in its cytoplasmic coding sequences. By contrast, the insertionally activated c-erbB, a variant oncogene, which encodes a product that also lacks the ligand-binding domain but is otherwise unaltered in its cytoplasmic coding sequences, is capable of inducing leukemia but cannot induce sarcomas. In this report, we show that the critical changes for activating the sarcomagenic potential displayed by v-erbB R are two point mutations within the tyrosine kinase domain and an internal deletion of 21 amino acids in the carboxyl-terminal regulatory domain. The removal of the carboxyl-terminal autophosphorylation sites is not obligatory. These activating mutations (Arg-263 to His, Ile-384 to Ser, and the deletion of residues 494 to 514), when introduced singly into the insertionally activated c-erbB, all dramatically increase fibroblast-transforming potential. Arg-263 resides near the highly conserved HRD motif of the kinase domain, and its mutation to His increases the autophosphorylation activity. The other two mutations do not alter the intrinsic kinase activity and presumably affect other aspects of the receptor involved in growth signaling. Therefore, the high transforming potential of v-erbB R is a consequence of synergism among multiple activating mutations.
Reassessment of the v-fms sequence: threonine phosphorylation of the COOH-terminal domain
Authors: Smola U , Hennig D , Hadwiger-Fangmeier A , Schütz B , Pfaff E , Niemann H , Tamura T
Published: January 2020

The v-fms oncogene product of the McDonough strain of feline sarcoma virus is a member of the receptor tyrosine kinase family. Its cellular counterpart, the c-fms product, is the receptor for colony-stimulating factor 1 (CSF-1) of macrophages. We have reanalyzed the v-fms gene by direct sequencing of a biologically active clone. An additional A nucleotide was detected in position 2810 of the published v-fms sequence. The frameshift changed the COOH-terminal sequence of the v-fms protein from -R-937-G-P-P-L-COOH to -Q-937-R-T-P-P-V-A-R-COOH. Antibodies against a synthetic peptide representing this new sequence precipitated the v-fms proteins from transformed NRK cells as well as from feline sarcoma virus (McDonough)-infected feline fibroblasts. We show by tryptic peptide mapping that threonine 939 present in the new sequence is phosphorylated by a yet unknown serine/threonine kinase in vivo. In chicken fibroblasts expressing the v-fms gene, this phosphorylation clearly depended on the addition of exogenous CSF-1. Furthermore, addition of CSF-1 appeared to activate the serine/threonine kinase, as judged by phosphorylation of the synthetic peptide QRTPPVAR.
Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo
Authors: Gilligan K J , Rajadurai P , Lin J C , Busson P , Abdel-Hamid M , Prasad U , Tursz T , Raab-Traub N
Published: January 2020

A family of mRNAs that are transcribed rightward through the BamHI A fragment have been detected in C15, a nasopharyngeal carcinoma (NPC) which has been passaged in nude mice. Northern (RNA) blot hybridizations indicate that these RNAs are also expressed in three other NPCs which have been established in nude mice and in an NPC obtained at biopsy. Moreover, hybridization in situ detected transcription from BamHI A in 12 NPCs and 1 Epstein-Barr virus (EBV)-containing carcinoma of the parotid gland. In each case, transcription was detected in all of the malignant epithelial cells. Transcription was not detected in two cases of EBV-positive lymphoma biopsies by in situ hybridization nor in latently infected EBV-positive lymphoblastoid cell lines by Northern blot hybridization. The consistent transcription of these sequences in latently infected epithelial malignancy but not in lymphoid cells suggests that this viral function is associated with latent EBV infection of epithelial cells. Sequence analysis of a cDNA synthesized from the C15 tumor, representing the 3' end of BamHI A messenger RNA, revealed an open reading frame (ORF). Translation of this ORF in vitro produced several peptides that were immunoprecipitated with antisera from patients with NPC. The detection of antibodies to the protein encoded by the ORF present in the BamHI A cDNA indicates that BamHI A encodes a protein which is expressed in vivo and is antigenic.
Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication
Authors: Camp H S , Coussens P M , Silva R F
Published: January 2020

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.
The 5' end of coronavirus minus-strand RNAs contains a short poly(U) tract
Authors: Hofmann M A , Brain D A
Published: January 2020

A radiolabeled oligodeoxynucleotide primer that anneals near the common 5' end of bovine coronavirus minus-strand RNAs was extended with reverse transcriptase, and a major product suggesting poly(U) tracts of 8 to 20 nucleotides was found. The extended primer molecules were ligated head to tail, amplified by the polymerase chain reaction, cloned, and sequenced, and poly(U) tracts of 9 to 26 nucleotides were found. Poly(A) tails of 100 to 130 nucleotides on the 3' end of coronavirus plus-strand mRNAs and genome must, therefore, be generated by a mechanism that uses only a short poly(U) template. This pattern contrasts with that of other cytoplasmic, polyadenylated, plus-strand animal RNA viruses which utilize a full-length poly(U) template for poly(A) synthesis.
Use of recombinant retroviruses to characterize the activity of antiretroviral compounds
Authors: Stair R K , Nelson C J , Mellors J W
Published: January 2020

This report describes the use of a recombinant murine retrovirus encoding beta-galactosidase (PLJ beta-gal retrovirus) to study the antiretroviral activity of zidovudine (AZT) and other nucleoside analogs. The PLJ beta-gal virus permits the rapid and unequivocal identification of individual virus-infected cells arising from a single cycle of viral replication. With this model system, AZT is shown to completely and irreversibly prevent retrovirus infection of proliferating cell lines as measured by a lack of reporter gene expression. On the other hand, AZT is less effective in protecting growth-arrested cells from retroviral infection. Recombinant retroviruses such as the PLJ beta-gal virus are potentially useful reagents for the identification and characterization of antiretroviral compounds.
Role of conserved gp41 cysteine residues in the processing of human immunodeficiency virus envelope precursor and viral infectivity
Authors: Syu W J , Lee W R , Du B , Yu Q C , Essex M , Lee T H
Published: January 2020

All animal retroviruses whose nucleotide sequences have been determined contain two or three closely spaced cysteine residues in the extracellular domain of the env-encoded transmembrane protein. Using human immunodeficiency virus type 1 gp41 as a working model, the functional significance of these highly conserved cysteines was investigated. We report here that substituting the two conserved cysteine residues in this domain of gp41 with glycine residues resulted in the loss of viral infectivity, which could be attributed to severe impairment in the processing of gp160 precursor to gp120.
The adenovirus E1A transforming protein activates the proliferating cell nuclear antigen promoter via an activating transcription factor site
Authors: Morris G F , Mathews M B
Published: January 2020

The transforming region of adenovirus (E1) stimulates expression of a reporter construct linked to the promoter for the human proliferating cell nuclear antigen (PCNA) gene in a cotransfection assay (G. F. Morris and M. B. Mathews, J. Biol. Chem. 264:13856-13864, 1989). The major products of the E1 region were assessed individually for their contribution to transactivation of the PCNA promoter. The E1A 13S and 12S products and the E1B 19-kDa product elevated expression from the PCNA promoter, whereas the E1B 55-kDa product did not. Induction of the PCNA promoter by E1A differed from transcriptional activation of the adenovirus E3 promoter in that the PCNA promoter is activated by the E1A 12S product whereas the E3 promoter is repressed; furthermore, the PCNA promoter is activated upon E1A overexpression, whereas the E3 promoter responds less well to high amounts of E1A. A site for the activating transcription factor ATF located approximately 50 nucleotides upstream from the transcription initiation site in the PCNA promoter mediates a positive response to the E1A 12S and 13S products.
Mutant varicella-zoster virus thymidine kinase: correlation of clinical resistance and enzyme impairment
Authors: Roberts G B , Fyfe J A , Gaillard R K , Short S A
Published: January 2020

Varicella-zoster virus (VZV) encodes a thymidine kinase (EC 2.7.2.21) which phosphorylates several antiviral nucleoside analogs, including acyclovir (ACV). A mutation in the VZV thymidine kinase coding sequence, resulting in an arginine-to-glutamine substitution at amino acid residue 130 (R130Q), is associated with clinical resistance to ACV. We have expressed the wild-type and the mutant enzymes in bacteria and have studied the kinetic characteristics of the purified enzymes. The arginine-to-glutamine substitution resulted in decreased catalytic activity and altered substrate specificity. The most striking effect was a decrease in the rates of nucleoside phosphorylation to less than 2% of the rates with the wild-type enzyme. This was accompanied by increased apparent Km values for thymidine and deoxycytidine. ACV was not detectably phosphorylated by the R130Q enzyme but still competed with thymidine for the enzyme. The inability of the R130Q enzyme to catalyze the phosphorylation of ACV correlates with resistance to ACV noted with a clinical isolate of VZV.