bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that a cell cycle regulator, E2F1, down-regulates transcriptional activity of the human immunodeficiency virus type 1 promoter
Authors: Kundu M , Srinivasan A , Pomerantz R J , Khalili K
Published: January 2020

Proliferation of eukaryotic cells is orchestrated by a series of cellular proteins which participate in various stages of the cell cycle to guide the cell through mitosis. Some of these proteins, including E2F1, play a critical role in G1 and S phases by coordinately regulating expression of several important cell cycle-associated genes. On the basis of recent observations indicating a block in human immunodeficiency virus type 1 (HIV-1) replication in cells arrested in G1/S phase of the cell cycle, we sought to evaluate the regulatory action of E2F1 on transcription from the HIV-1 long terminal repeat (LTR). Results from transient transfection of cells with an E2F1 expression plasmid indicated that E2F1 has the ability to suppress basal transcriptional activity of the LTR and to diminish the extent of the Tat-induced activation of the viral promoter. Deletion analysis of the HIV-1 LTR in transfection studies revealed the presence of two major elements responsive to E2F1 repression located distally (-454 to -381) and proximally (-117 to -80) with respect to the +1 transcription start site. E2F1-mediated suppression of LTR activity was observed in a wide range of human cell lines. Expression of E2F1 by a transgene showed an inhibitory effect on the levels of reverse transcriptase activity obtained upon introduction of the proviral genome into cells. The data presented in this study suggest that cellular regulatory proteins involved in the progression of cells through the mitotic cycle could play crucial roles in determining the efficiency of HIV-1 replication during the various stages of infection. The possible roles of these factors in viral latency and activation are discussed.
Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation
Authors: Yao X J , Subbramanian R A , Rougeau N , Boisvert F , Bergeron D , Cohen E A
Published: January 2020

The Vpr gene product of human immunodeficiency virus type 1 is a virion-associated protein that is important for efficient viral replication in nondividing cells such as macrophages. At the cellular level, Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Incorporation of Vpr into viral particles requires a determinant within the p6 domain of the Gag precursor polyprotein Pr55gag. In the present study, we have used site-directed mutagenesis to identify a domain(s) of Vpr involved in virion incorporation and nuclear localization. Truncations of the carboxyl (C)-terminal domain, rich in basic residues, resulted in a less stable Vpr protein and in the impairment of both virion incorporation and nuclear localization. However, introduction of individual substitution mutations in this region did not impair Vpr nuclear localization and virion incorporation, suggesting that this region is necessary for the stability and/or optimal protein conformation relevant to these Vpr functions. In contrast, the substitution mutations within the amino (N)-terminal region of Vpr that is predicted to adopt an alpha-helical structure (extending from amino acids 16 to 34) impaired both virion incorporation and nuclear localization, suggesting that this structure may play a pivotal role in modulating both of these biological properties. These results are in agreement with a recent study showing that the introduction of proline residues in this predicted alpha-helical region abolished Vpr virion incorporation, presumably by disrupting this secondary structure (S. Mahalingam, S. A. Khan, R. Murali, M. A. Jabbar, C. E. Monken, R. G. Collman, and A. Srinivasan, Proc. Natl. Acad. Sci. USA 92:3794-3798, 1995). Interestingly, our results show that two Vpr mutants harboring single amino acid substitutions (L to F at position 23 [L23F] and A30F) on the hydrophobic face of the predicted helix coded for relatively stable proteins that retained their ability to translocate to the nucleus but exhibited dramatic reduction in Vpr incorporation, suggesting that this hydrophobic face might mediate protein-protein interactions required for Vpr virion incorporation but not nuclear localization. Furthermore, a single mutation (E25K) located on the hydrophilic face of this predicted alpha-helical structure affected not only virion incorporation but also nuclear localization of Vpr. The differential impairment of Vpr nuclear localization and virion incorporation by mutations in the predicted N-terminal alpha-helical region suggests that this region of Vpr plays a role in both of these biological functions of Vpr.
Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus
Authors: Ericsson M , Cudmore S , Shuman S , Condit R C , Griffiths G , Locker J K
Published: January 2020

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.
Enhanced splicing of nonconsensus 3' splice sites late during adenovirus infection
Authors: Mühlemann O , Kreivi J P , Akusjärvi G
Published: January 2020

The adenovirus major late transcription unit is an example of an alternatively spliced gene, in which a common 5' splice site can be spliced to more than 15 different 3' splice sites. Here we show that the specificity in 3' splice site recognition changes during virus infection, such that 3' splice sites with long consensus-type polypyrimidine tracts are repressed while 3' splice sites with short atypical polypyrimidine tracts, which bind U2AF65K inefficiently, are enhanced in splicing in late virus-infected nuclear extracts. On the basis of these experiments, we discuss a mechanism that helps to explain how the complex pattern of major late mRNAs is produced late during virus infection.
Isolation of a highly cytopathic lentivirus from a nondomestic cat
Authors: Barr M C , Zou L , Holzschu D L , Phillips L , Scott F W , Casey J W , Avery R J
Published: January 2020

A feline immunodeficiency virus-like virus (FIV-Oma) isolated from a Pallas' cat (Otocolobus manul) is highly cytopathic in CrFK cells, in contrast to the chronic, noncytolytic infection established by an FIV isolate from a domestic cat (FIV-Fca). The virions have typical lentivirus morphology, density, and magnesium-dependent reverse transcriptase activity. The major core protein is antigenically cross-reactive with that of FIV-Fca; however, FIV-Oma transcripts do not cross-hybridize with FIV-Fca. A conserved region of the FIV-Oma pol gene has 76 to 80% nucleic acid identify with the corresponding pol regions of other feline lentiviruses and 64 to 69% identity with those of human, ovine, and equine lentiviruses.
Function of a 5'-end genomic RNA mutation that evolves during persistent mouse hepatitis virus infection in vitro
Authors: Chen W , Baric R S
Published: January 2020

Persistently infected cultures of DBT cells were established with mouse hepatitis virus strain A59 (MHV-A59), and the evolution of the MHV leader RNA and 5' end of the genome was studied through 119 days postinfection. Sequence analysis of independent clones demonstrated an overall mutation frequency approaching 1.2 x 10(-3) to 6.7 x 10(-3). The rate of fixation of mutations was about 1.2 x 10(-5) to 7.6 x 10(-5) per nucleotide (nt) per day. In contrast to finding in bovine coronavirus, the MHV leader RNA sequences were extremely stable and did not evolve significantly during persistent infection. Rather, a 5' untranslated region (UTR) A-to-G mutation at nt 77 in the genomic RNA emerged by day 56 and accumulated until 50 to 80% of the genome-length molecules retained the mutation by 119 days postinfection. Although other 5'-end mutations were noted, only the nt 77 mutation was significantly associated with viral persistence in vitro. Mutations were also found in the 5' end of the p28 coding region, but no specific alterations accumulated in genome-length molecules through 119 days postinfection. The 5' UTR nt 77 mutation resulted in an 18-amino-acid open reading frame (ORF) upstream of the ORF 1a AUG start site. By in vitro translation assays, the small ORF was not translated into detectable product but the mutation significantly enhanced translation of the downstream p28 ORF about 2.5-fold. Variant viruses, containing either the nt 77 A-to-G mutation (V16-ATG+) or wild-type sequences at this locus (V1-ATG-), were isolated at 119 days postinfection. The variant viruses replicated more efficiently than wild-type virus and were extremely cytolytic in DBT cells, suggesting that the A-to-G mutation did not encode a nonlytic or attenuated phenotype. Consistent with the in vitro translation results, a significant increase (approximately 3.5-fold) in p28 expression was also observed with the mutant virus (V16-ATG+) in DBT cells compared with that in wild-type controls. These data indicate that MHV persistence was significantly associated with mutation and evolution in the 5'-end UTR which enhanced the translation of the ORF 1a and potentially ORF 1b polyproteins which function in virus transcription and replication.
Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin
Authors: Justewicz D M , Morin M J , Robinson H L , Webster R G
Published: January 2020

Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.
The dual-specificity phosphatase encoded by vaccinia virus, VH1, is essential for viral transcription in vivo and in vitro
Authors: Liu K , Lemon B , Traktman P
Published: January 2020

The genetic complexity of vaccinia virus is such that as well as encoding its own transcription and replication machinery, it encodes two protein kinases and a protein phosphatase. The latter enzyme, designated VH1, is a prototype for the dual-specificity class of phosphatases. Here we report that the H1 phosphatase is encapsidated within vaccinia virions and describe the construction of a viral recombinant in which expression of the H1 gene is regulated by the presence or absence of isopropylthiogalactopyranoside (IPTG) in the culture medium. When expression of H1 is repressed, the number of viral particles produced is not compromised but the fraction of these particles which is infectious is significantly reduced. The lack of infectivity of the H1-deficient particles is specifically correlated with their inability to direct the transcription of early genes either in vitro or in vivo. A proximal role for the viral phosphatase in regulating the onset of viral gene expression is implied. Prominent among the encapsidated proteins found to be hyperphosphorylated in H1-deficient virions is the 11-kDa product of the F18 gene; this protein is the major DNA-binding component of the viral nucleoprotein complex. The ability of recombinant H1 phosphatase to reverse this hyperphosphorylation in permeabilized virions strengthens the conclusion that the F18 protein is a bona fide substrate for the H1 phosphatase.
Distribution of baboon endogenous virus among species of African monkeys suggests multiple ancient cross-species transmissions in shared habitats
Authors: van der Kuyl A C , Dekker J T , Goudsmit J
Published: January 2020

PCR amplification of baboon endogenous virus (BaEV) long terminal repeat, reverse transcriptase gene, and env fragments from 24 different species of African monkeys indicates that BaEV is less widespread than was formerly thought. Instead of being present in every species of African primates, BaEV can be found only in baboons, geladas, and mangabeys (all belonging to the Papionini tribe) and in African green monkey (Cercopithecus aethiops)subspecies. BaEV, which can be activated from baboon and gelada tissues, was most likely introduced in the germ line only recently (less than a few million years ago) and has not been inherited from a common ancestor of all extant African monkeys. Neighbor-joining and maximum-likelihood analyses of the sequences obtained showed that two distinct virus clusters can be distinguished: the first containing baboon, gelada, and African green monkey BaEV sequences and the second consisting of mandrill and mangabey BaEV sequences. This viral evolutionary tree does not follow host phylogeny, indicating the cross-species transmissions and multiple germ line fixations of the virus must have occurred in the past. BaEV sequences are found in monkeys inhabiting savannas (baboons, geladas, and African green monkeys) as well as forests (managabeys and mandrills) and cluster according to the habitats of their hosts, providing evidence for cross-species transmission in shared habitats.
E1A promotes association between p300 and pRB in multimeric complexes required for normal biological activity
Authors: Wang H G , Moran E , Yaciuk P
Published: January 2020

The oncogenes of the small DNA tumor viruses encode transforming proteins with multiple domains that influence the cell cycle and aspects of the transformed phenotype. Like other gene products of this type, the adenovirus E1A proteins influence the cell by binding to specific cell growth control proteins. These include members of the retinoblastoma gene product (pRB) family, which are bound by the E1A region 2-specific site, and p300, which is bound at the E1A amino terminus. Binding at these two sites is largely independent, and discrete transcription-regulating functions remain intact in E1A products when only one or the other binding site is functional. In this report, immunoprecipitation with p300 antibodies reveals the presence of the pRB family proteins in p300 complexes when E1A is expressed in host cells, indicating that E1A can mediate physical contact between p300 and the pRB-related proteins. The ability of E1A to induce proliferation efficiently in quiescent primary cells correlates closely with the ability to bind p300 and individual members of the pRB family simultaneously in multimeric complexes, even though the E1A active sites can bind their target proteins efficiently when separated on different molecules. Conservation of a spacer region between the two binding sites that is required for simultaneous binding and efficient induction of proliferation supports the concept that the E1A protein structure has evolved to facilitate simultaneous binding. These results indicate that the E1A proteins are designed not merely to sequester these cellular products, but also to bring them into proximal association with each other in biologically significant complexes.
Genetic and structural analysis of a virulence determinant in polyomavirus VP1
Authors: Bauer P H , Bronson R T , Fung S C , Freund R , Stehle T , Harrison S C , Benjamin T L
Published: January 2020

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.
Genotypic selection of simian immunodeficiency virus in macaque infants infected transplacentally
Authors: Amedee A M , Lacour N , Gierman J L , Martin L N , Clements J E , Bohm R , Harrison R M , Murphey-Corb M
Published: January 2020

To understand viral and host factors that contribute to transplacental transmission of human immunodeficiency virus, we developed an animal model using pregnant female macaques infected with simian immunodeficiency virus (SIV). Pregnant females were inoculated intravenously during midgestation with either a well-characterized primary isolate of SIV (SIV/DeltaB670) or a combination of SIV/DeltaB670 and the macrophage-tropic molecular clone SIV/17E-Fr. The viral genetic diversity in five infected female macaques and their in utero-infected infants was analyzed. All of the mothers harbored a genetically diverse virus population at parturition, whereas a single genotype from the maternal quasispecies was identified in the infants at birth. Only one of two variants was found in the infants: SIV/17E-Fr (two cases) or a genotype contained within the SIV/DeltaB670 quasispecies (three cases). The macrophage-tropic properties of both transmitted genotypes were suggested by productive replication in primary rhesus macrophage cultures in vitro and the clonal presence in central nervous system tissue of infected monkeys with encephalitis. These observations provide compelling evidence for both genotypic and phenotypic selection in transplacental transmission of SIV and suggest a critical role for macrophages in fetal infection in utero.
Expression of ecotropic murine leukemia virus in the brains of C58/M, DBA2/J, and in utero-infected CE/J mice
Authors: Anderson G W , Plagemann P G
Published: January 2020

In C58 and AKR mice, endogenous N-tropic, ecotropic murine leukemia virus (MuLV) proviruses become activated in rare cells during embryogenesis. Resultant replication-competent progeny viruses then actively infect a large number of cells throughout the fetus, including cells in the developing central nervous system. By in situ hybridization analyses, we have assessed the presence of ecotropic MuLV RNA in the brains of C58 mice as a function of age. Only a few ecotropic MuLV-positive cells were observed in weanling mice, but the number of positive cells in the brain increased progressively with increasing age of the mice. Throughout the lives of the mice, the ecotropic MuLV RNA-positive cells were primarily located in well-defined white-matter tracts of the brain (commissura anterior, corpus callosum, fimbria hippocampi, optical tract, and striatum) and of the spinal cord. Cells of the subventricular zone also expressed ecotropic MuLV RNA, and in older mice a small number of positive cells were present in the grey matter. Infection of endogenous ecotropic MuLV provirus-less CE/J mice in utero with ecotropic MuLV clone AKR-623 resulted in the extensive infection of brain cells. The regional distribution of ecotropic MuLV RNA-containing cells was the same as observed in the brains of C58 mice, in which cells became infected by endogenously activated virus, but the number of positive cells was higher.
Molecular linkage of hantavirus pulmonary syndrome to the white-footed mouse, Peromyscus leucopus: genetic characterization of the M genome of New York virus
Authors: Hjelle B , Lee S W , Song W , Torrez-Martinez N , Song J W , Yanagihara R , Gavrilovskaya I , Mackow E R
Published: January 2020

The complete M segment sequences of hantaviruses amplified from tissues of a patient with hantavirus pulmonary syndrome in the northeastern United States and from white-footed mice, Peromyscus leucopus, from New York were 99% identical and differed from those of Four Corners virus by 23%. The serum of this patient failed to recognize a conserved, immunodominant epitope of the Four Corners virus G1 glycoprotein. Collectively, these findings indicate that P. leucopus harbors a genetically and antigenically distinct hantavirus that causes hantavirus pulmonary syndrome.
Importance of cysteines in the LDLR-related domain of the subgroup A avian leukosis and sarcoma virus receptor for viral entry
Authors: Bélanger C , Zingler K , Young J A
Published: January 2020

The extracellular domain of the subgroup A avian sarcoma and leukemia virus (ALSV-A) receptor contains a region that is related in sequence to the ligand-binding motifs of the low-density lipoprotein receptor (LDLR). This domain contains six cysteines that are highly conserved between different members of the LDLR protein superfamily, and these residues are presumed to participate in intrachain disulfide bonds. To assess the importance of each cysteine in the ALSV-A receptor, individual or multiple cysteines were mutated to alanines and the altered receptors were tested for the ability to confer susceptibility to viral infection. Receptors bearing single mutations allowed subgroup A viral entry, albeit at less than wild-type levels. Receptors containing two or three substitutions were completely inactive if one of the changed residues was Cys-35 or Cys-50. Of the altered receptors tested, the only exception to this rule was a functional receptor which lacked both Cys-35 and Cys-50, an activity that was dependent on the presence of other cysteines in this protein. Most interestingly, a receptor containing both Cys-35 and Cys-50 but lacking the other four cysteines was completely functional. These results demonstrate the importance of Cys-35 and Cys-50 for viral entry mediated by the ALSV-A receptor and show that in the presence of these two residues, all of the other cysteines in this protein can be removed without loss of this function.
The dimerization/packaging sequence is dispensable for both the formation of high-molecular-weight RNA complexes within retroviral particles and the synthesis of proviruses of normal structure
Authors: Tchénio T , Heidmann T
Published: January 2020

Retroviral particles contain a dimer of two genomic RNA molecules, linked by noncovalent intermolecular bonds. Studies by electron microscopy of viral RNA extracted from virions as well as in vitro studies have implicated a sequence, designated the dimer linkage sequence (DLS), in the dimerization process. The DLS has been localized within a short region encompassing the psi packaging sequence, between nucleotides 212 and 563 for the Moloney murine leukemia retrovirus (MoMLV) RNA. In this report, we show that viral RNAs lacking both the DLS and psi packaging sequences--and even an RNA lacking the first 6,537 nucleotides of MoMLV--can assemble within retroviral particles as high-molecular-weight, slow-migrating, heat-sensitive complexes closely related to those observed for wild-type viral RNAs. Furthermore, we show that proviruses of normal structure are generated upon infection of test cells with retroviral particles which contain the DLS/psi-deleted viral RNAs. These observations demonstrate that the DLS and psi packaging sequences are not essential in cis to form a functional RNA complex for reverse transcription and integration.
Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation
Authors: Shih C M , Chen C M , Chen S Y , Lee Y H
Published: January 2020

We previously demonstrated that the core protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV core protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV core protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion protein expression system and in vitro transcription and translation system, the phosphorylation of HCV core protein was carried out by protein kinase A (PKA) and protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in core protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV core protein was essential for the suppressive activity of HCV core protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV core protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV core protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV core protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV core protein is a phospho-protein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue.(ABSTRACT TRUNCATED AT 250 WORDS)
A minor tyrosine phosphorylation site located within the CAIN domain plays a critical role in regulating tissue-specific transformation by erbB kinase
Authors: Chang C M , Shu H K , Ravi L , Pelley R J , Shu H , Kung H J
Published: January 2020

Avian c-erbB encodes a protein that is homologous to the human epidermal growth factor receptor. Truncation of the amino-terminal, ligand-binding domain of this receptor results in an oncogene product which is a potent inducing agent for erythroleukemias but not fibrosarcomas in chickens. Here we show that mutation of a single tyrosine residue, p5, in the carboxyl terminus of the erbB oncogene product allows it to become sarcomagenic in vivo and to transform fibroblasts in vitro. Mutations of other autophosphorylation sites do not generate comparable effects. The increased transforming activity of the p5 mutant is accompanied by an elevated level of mitogen-activated protein kinase phosphorylation. By analogy to the human epidermal growth factor receptor, p5 is a minor autophosphorylation site and is located in a domain known to be involved in regulating calcium influx and receptor internalization (CAIN domain). This area of the erbB product has been found to be repeatedly deleted in various sarcomagenic avian erythroblastosis virus isolates. We precisely deleted the CAIN domain and also made point mutations of the acidic residues within the CAIN domain. In both cases, fibroblast-transforming potential is activated. We interpret these data to mean that p5 and its surrounding region negatively regulate fibroblast-transforming and sarcomagenic potential. To our knowledge, this represents the first point mutation of an autophosphorylation site that activates erbB oncogenicity.
Evidence for coinfection by multiple strains of human immunodeficiency virus type 1 subtype B in an acute seroconvertor
Authors: Zhu T , Wang N , Carr A , Wolinsky S , Ho D D
Published: January 2020

Sequences encoding the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) were amplified by PCR from plasma and peripheral blood mononuclear cells obtained at four time points from an acute seroconvertor. Genetic analyses, including nucleotide sequencing and heteroduplex mobility studies, showed that the patient harbored three distinct populations of HIV-1 clade B envelope sequences, with nucleotide distances ranging from 9.2 to 17.2%. One population of sequences was clearly distinguishable from the others on the basis of phylogenetic analysis. In addition, sequences suggesting recombination between two of the three distinct viral populations were also found. This case of acute seroconversion provides clear and conclusive evidence that coinfection by multiple HIV-1 strains can indeed occur in vivo.
Phylogenetic relationship and geographic distribution of multiple human T-cell lymphotropic virus type II subtypes
Authors: Switzer W M , Pieniazek D , Swanson P , Samdal H H , Soriano V , Khabbaz R F , Kaplan J E , Lal R B , Heneine W
Published: January 2020

The current env-based subtyping of human T-cell lymphotropic virus type II (HTLV-II) identifies only two heterogenetic groups, HTLV-IIa and HTLV-IIb. To better understand the genetic diversity and phylogeny of HTLV-II, we examined the most divergent genomic region of HTLV-II, the long terminal repeat, by using restriction fragment length polymorphism (RFLP) and sequence analysis. Long terminal repeat sequences were amplified from peripheral blood mononuclear cells by PCR and digested with seven restriction endonucleases that differentiated HTLV-II into five HTLV-IIa (IIa0 to IIa4) and six HTLV-IIb (IIb0 to IIb5) restriction types, with HTLV-IIa0 and HTLV-IIb0 being prototypes for the MoT and NRA isolates, respectively. We examined 169 HTLV-II-infected samples, including 123 from blood donors and intravenous drug users (IDU) from the Americas, 16 from IDU from Europe, and 30 from Amerindians. Of the 169 samples, 109 (64.5%) were categorized as HTLV-IIa and 60 (35.5%) were categorized as HTLV-IIb. The predominant restriction types seen among the U.S. blood donors and U.S. IDU were IIa0 (68.7%) and IIb4 (10.4%). Four Spanish and seven Italian samples were IIb4, while five Norwegian samples were IIa2. Twelve Guaymi and all ten Seminole samples were single restriction types (IIb1 and IIb5, respectively), whereas the two Navajo and six Pueblo samples had a mixture of restriction types IIa0, IIa4, and IIb5. Of the HTLV-IIb restriction types observed in the U.S. non-Indians, 42.8% appear to have originated from the North Amerindian (IIb5), while 57.2% were similar to the European IIb4 restriction type. Sequences of 15 selected HTLV-II samples were determined and phylogenetically compared with 7 previously published HTLV-II LTR sequences. The derived topologies revealed three HTLV-IIa phylogroups (A-I to A-III) and four HTLV-IIb phylogroups (B-I to B-IV). Furthermore, the HTLV-IIa phylogroups appear to have evolved from the HTLV-IIb phylogroups. In the HTLV-IIa cluster, a Navajo (A-I) and a Brazilian (A-II) sequence formed separate phylogroups, while the remaining IIa sequences formed a single phylogroup (A-III). The four HTLV-IIb phylogroups were represented predominantly by a New York IDU (B-I), European IDU (B-II), North Amerindian and NRA (B-III), and Central Guaymi Indian (B-IV) sequence(s). Comparison of the phylogenetic data with the RFLP results revealed that results of the two methods correlated completely, demonstrating the ability of the RFLP method to predict the phylogroup of HTLV-II-infected samples accurately and quickly. GENBANK/U10258
Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release, and infectivity
Authors: Reicin A S , Paik S , Berkowitz R D , Luban J , Lowy I , Goff S P
Published: January 2020

The phenotypes of a series of mutant human immunodeficiency virus type 1 proviruses with linker insertion and deletion mutations within the gag coding region were characterized. These mutants were tested for their ability to make and release viral particles in COS7 cells and for their viability in vivo. Of the 12 mutant proviruses, 4 did not make extracellular virion particles when transfected into COS7 cells. All four of these mutants had mutations in the C-terminal domain of CA. These mutants appeared to have defects both in the ability to accumulate high-molecular-weight intracellular structures containing Gag and Pol products and in the ability to release virion particles. Seven of the mutant proviruses retained the ability to make, release, and process virion particles from COS7 cells. These particles contained the Env glycoprotein, viral genomic RNA, and the mature products of the Gag and Gag-Pol polyproteins, yet they were noninfectious or poorly infectious. The defect in these mutants appears to be in one of the early steps of the viral life cycle. Thus, multiple regions throughout Gag appear to be important in mediating the early steps of the viral life cycle.
Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH
Authors: Allison S L , Schalich J , Stiasny K , Mandl C W , Kunz C , Heinz F X
Published: January 2020

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.
Identification of the murine coronavirus p28 cleavage site
Authors: Hughes S A , Bonilla P J , Weiss S R
Published: January 2020

Mouse hepatitis virus strain A59 encodes a papain-like cysteine proteinase (PLP-1) that, during translation of ORF1a, cleaves p28 from the amino terminus of the growing polypeptide chain. In order to determine the amino acid sequences surrounding the p28 cleavage site, the first 4.6 kb of murine hepatitis virus strain A59 ORF1a was expressed in a cell-free transcription-translation system. Amino-terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly-247 and Val-248. Site-directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg-246 (P2) and Gly-247 (P1) nearly eliminated cleavage of p28. Single-amino-acid substitutions of other residues between P7 and P2' were generally permissive for cleavage, although a few changes did greatly reduce proteolysis. The relationship between the p28 cleavage site and other viral and cellular papain proteinase cleavage sites is discussed.
The T-cell-independent role of gamma interferon and tumor necrosis factor alpha in macrophage activation during murine cytomegalovirus and herpes simplex virus infections
Authors: Heise M T , Virgin H W
Published: January 2020

We defined the normal and innate (without functional B or T cells) inflammatory response to infection with mouse cytomegalovirus (MCMV) or herpes simplex virus (HSV). Intraperitoneal infection with MCMV or HSV induced an inflammatory infiltrate consisting largely of macrophages (M phi) in both normal CB17 and severe combined immunodeficient (SCID) mice (lacking functional B or T cells). M phi from infected mice were activated as shown by (i) spread morphology, (ii) increased expression of major histocompatibility complex (MHC) class II, MHC class I, and intercellular adhesion molecule-1 molecules, and (iii) downregulation of M phi-specific cell surface protein F4/80. In vivo administration of neutralizing antibodies specific for gamma interferon (IFN gamma) or tumor necrosis factor alpha (TNF alpha) inhibited MHC class II induction on infiltrating M phi in both normal and CB17 SCID mice. Anti-TNF alpha decreased the number of M phi in virus-induced inflammatory exudates. The MCMV titer increased in the spleen and liver of IFN gamma-depleted SCID mice, while TNF alpha depletion increased only splenic titers. MCMV-induced pathology was also increased in spleens of IFN gamma- and TNF alpha-depleted SCID mice. We conclude that (i) M phi activation is a prominent part of inflammatory responses to herpesvirus infection and (ii) IFN gamma and TNF alpha play a critical role in both virus-induced M phi activation and control of herpesvirus growth independent of T and B cells. This suggests that IFN gamma- and TNF alpha-mediated M phi activation is an important aspect of innate immunity to viral infection. As the M phi may be involved in MCMV latency, IFN gamma- and TNF alpha-dependent M phi activation during primary infection may be relevant to establishment of viral latency.
Characterization of nuclear protein binding to a site in the long terminal repeat of a murine leukemia virus: comparison with the NFAT complex
Authors: Yoshimura F K , Diem K
Published: January 2020

We previously identified a protein-binding site (MLPal) that is located downstream of the enhancer element in the long terminal repeat (LTR) of a mink cell focusing-forming (MCF) murine leukemia virus (F. K. Yoshimura, K. Diem, H. Chen, and J. Tupper, J. Virol. 67:2298-2304, 1993). We determined that the MLPal site regulates transcription specifically in T cells and affects the lymphomagenicity of the MCF isolate 13 murine leukemia virus with a single enhancer repeat in its LTR. In this report, we present evidence that two different proteins, a T-cell-specific protein and a ubiquitous protein, bind the MLPal site in a sequence-specific manner. By mutational analysis, we determined that the T-cell-specific and the ubiquitous proteins require different nucleotides in the MLPal sequence for DNA binding. By competitive electrophoretic mobility shift assays, we demonstrated that the T-cell-specific protein that binds MLPal is identical or similar to a protein from nonactivable T cells that interacts with the binding site of the nuclear factor of activated T cells (NFAT). Unlike the NFAT-binding site, however, the MLPal site does not bind proteins that are inducible by T-cell activation. We observed that the MLPal sequence is conserved in the LTRs of other mammalian retroviruses that cause T-cell diseases. Furthermore, the MLPal sequence is present in the transcriptional regulatory regions of cellular genes that either are expressed specifically in T cells or are commonly rearranged by provirus integration in thymic lymphomas. Thus, the MLPal-binding proteins may play a role in the transcriptional regulation not only of the MCF virus LTR but also of cellular genes involved in T-cell development.
Cloning and characterization of herpes simplex virus type 1 oriL: comparison of replication and protein-DNA complex formation by oriL and oriS
Authors: Hardwicke M A , Schaffer P A
Published: January 2020

The herpes simplex virus type 1 genome contains three origins of DNA replication: two copies of oriS and one copy of oriL. Although oriS has been characterized extensively, characterization of oriL has been severely limited by the inability to amplify oriL sequences in an undeleted form in Escherichia coli. We report the successful cloning of intact oriL sequences in an E. coli strain, SURE, which contains mutations in a series of genes involved in independent DNA repair pathways shown to be important in the rearrangement and deletion of DNA containing irregular structures such as palindromes. The oriL-containing clones propagated in SURE cells contained no deletions, as determined by Southern blot hybridization and DNA sequence analysis, and were replication competent in transient DNA replication assays. Deletion of 400 bp of flanking sequences decreased the replication efficiency of oriL twofold in transient assays, demonstrating a role for flanking sequences in enhancing replication efficiency. Comparison of the replication efficiencies of an 822-bp oriS-containing plasmid and an 833-bp oriL-containing plasmid demonstrated that the kinetics of replication of the two plasmids were similar but that the oriL-containing plasmid replicated 60 to 70% as efficiently as the oriS-containing plasmid at both early and late times after infection with herpes simplex virus type 1. The virus-specified origin-binding protein (OBP) and a cellular factor(s) (OF-1) have been shown in gel mobility shift experiments to bind specific sequences in oriS (C.E. Dabrowski, P. Carmillo, and P.A. Schaffer, Mol. Cell. Biol. 14:2545-2555, 1994; C.E. Dabrowski and P.A. Schaffer, J. Virol. 65:3140-3150, 1991). Although the nucleotides required for the binding of OBP to OBP binding site I in oriL and oriS are the same, a single nucleotide difference distinguishes OBP binding site III in the two origins. The nucleotides adjacent to oriS sites I and III have been shown to be important for the binding of OF-1 to oriS site I. Several nucleotide differences exist in these sequences in oriL and oriS. Despite these minor nucleotide differences, the protein-DNA complexes that formed with oriL and oriS sites I and III were indistinguishable when extracts of infected and uninfected cells were used as the source of protein. Furthermore, the results of competition analysis suggest that the proteins involved in protein-DNA complex formation with sites I and III of the two origins are likely the same.
Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity
Authors: Fredericksen B L , Whitt M A
Published: January 2020

We have introduced amino acid substitutions into two regions of the extracellular domain of the vesicular stomatitis virus (VSV) glycoprotein (G protein) and examined the effect of these mutations on protein transport, low-pH-induced stability of G protein oligomers, and membrane fusion activity. We suggested previously that the region between amino acids 118 and 139 may be important for the membrane fusion activity of G protein, on the basis of the characterization of a fusion-defective G protein mutant (M. A. Whitt, P. Zagouras, B. Crise, and J. K. Rose, J. Virol. 64:4907-4913, 1990). It has also been postulated by others that this region as well as the region between amino acids 181 and 212 may constitute putative internal fusion domains of VSV G protein. In this report, we show that three different amino acids substitutions between residues 118 and 139 (G-124-->E, P-127-->D, and A-133-->K) either altered or abolished low-pH-dependent membrane fusion activity. In contrast, substitutions between residues 192 and 212 resulted either in G proteins that had wild-type fusion activity or in mutant proteins in which the mutation prevented transport of G protein to the cell surface. Two of the substitutions between residues 118 and 139 (G-124-->E and P-127-->D) resulted in G proteins that were fusion defective at pH 5.7, although syncytia were observed after cells were treated with fusion buffer at pH 5.5, albeit at levels significantly less than that induced by wild-type G protein. Interestingly, when either G-124-->E or P-127-->D was incorporated into tsO45 virions, the resulting particles were not infectious, presumably because the viral envelope was not able to fuse with the proper intracellular membrane. These results support the hypothesis that the region between amino acids 118 and 139 is important for the membrane fusion activity of VSV G protein and may constitute an internal fusion domain.