bluuebunny/crossref_metadata_2025_split huggingface dataset:
Relative Replicative Fitness of Zidovudine-Resistant Human Immunodeficiency Virus Type 1 Isolates In Vitro
Authors: Harrigan P. Richard , Bloor Stuart , Larder Brendan A. 
Published: December 2019

ABSTRACT

Replication of mixtures of two or more human immunodeficiency virus type 1 (HIV-1) variants would be expected to result in the eventual selection of the fittest virus due to Darwinian competition among the variants. The relative proportions of known HIV-1 variants (which may differ only by a single nucleotide from a standard “wild-type” virus, HIV-1
HXB2
) in mixed viral cultures were quantified by analysis of automated sequence signals of reverse transcriptase PCR products. With this method, the relative levels of replicative fitness of several zidovudine (3′-azidothymidine)-resistant HIV-1
HXB2
variants were estimated under controlled in vitro conditions by measuring the rate of change in the proportions of viral variants as they replicated in cell cultures both in the presence and in the absence of drug selection pressure. These variants were engineered to contain commonly observed zidovudine resistance mutations in the HIV-1 reverse transcriptase (M41L, K70R, T215Y, and M41L+T215Y). In the absence of zidovudine, all variants tested displayed reduced replicative fitness compared to wild-type HIV-1
HXB2
. The order of relative fitness was wild type > K70R ≫ T215Y = M41L+T215Y > M41L. Mixed cultures in the presence of zidovudine showed a dose-dependent selection pressure against the wild-type virus which varied according to the resistance profile of each virus. The information gathered from this approach provides insight into competition among multiple HIV-1 variants, which likely occurs in vivo with drug selection pressure, and may be applicable in more complex mathematical models for predicting the emergence of HIV-1 variants after the initiation of antiretroviral therapy.
Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase
Authors: Kau Jyh-Hwa , Ting Ling-Pai 
Published: December 2019

ABSTRACT
Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.
The Class II Membrane Glycoprotein G of Bovine Respiratory Syncytial Virus, Expressed from a Synthetic Open Reading Frame, Is Incorporated into Virions of Recombinant Bovine Herpesvirus 1
Authors: Kühnle Gisela , Heinze Astrid , Schmitt Jutta , Giesow Katrin , Taylor Geraldine , Morrison Ivan , Rijsewijk Frans A. M. , van Oirschot Jan T. , Keil Günther M. 
Published: December 2019

ABSTRACT

The bovine herpesvirus 1 (BHV-1) recombinants BHV-1/eG
ori
and BHV-1/eG
syn
were isolated after insertion of expression cassettes which contained either a genomic RNA-derived cDNA fragment (BHV-1/eG
ori
) or a modified, chemically synthesized open reading frame (ORF) (BHV-1/eG
syn
), which both encode the attachment glycoprotein G of bovine respiratory syncytial virus (BRSV), a class II membrane glycoprotein. Northern blot analyses and nuclear runoff transcription experiments indicated that transcripts encompassing the authentic BRSV G ORF were unstable in the nucleus of BHV-1/eG
ori
-infected cells. In contrast, high levels of BRSV G RNA were detected in BHV-1/eG
syn
-infected cells. Immunoblots showed that the BHV-1/eG
syn
-expressed BRSV G glycoprotein contains N- and O-linked carbohydrates and that it is incorporated into the membrane of infected cells and into the envelope of BHV-1/eG
syn
virions. The latter was also demonstrated by neutralization of BHV-1/eG
syn
infectivity by monoclonal antibodies or polyclonal anti-BRSV G antisera and complement. Our results show that expression of the BRSV G glycoprotein by BHV-1 was dependent on the modification of the BRSV G ORF and indicate that incorporation of class II membrane glycoproteins into BHV-1 virions does not necessarily require BHV-1-specific signals. This raises the possibility of targeting heterologous polypeptides to the viral envelope, which might enable the construction of BHV-1 recombinants with new biological properties and the development of improved BHV-1-based live and inactivated vector vaccines.
Epizootic Hemorrhagic Disease: Analysis of Tissues by Amplification and In Situ Hybridization Reveals Widespread Orbivirus Infection at Low Copy Numbers
Authors: Brodie Scott J. , Bardsley Katherine D. , Diem Kurt , Mecham James O. , Norelius Scott E. , Wilson William C. 
Published: December 2019

ABSTRACTA recent outbreak of hemorrhagic fever in wild ruminants in the northwest United States was characterized by rapid onset of fever, followed shortly thereafter by hemorrhage and death. As a result, a confirmed 1,000 white-tailed deer and pronghorn antelope died over the course of 3 months. Lesions were multisystemic and included severe edema, congestion, acute vascular necrosis, and hemorrhage. Animals that died with clinical signs and/or lesions consistent with hemorrhagic fever had antibody to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) by radioimmune precipitation but the antibody was limited exclusively to class immunoglobulin M. These findings, indicative of acute infection, were corroborated by the observation that numerous deer were found dead; however, clinically affected deer were rarely seen during the outbreak. Furthermore, only in animals with hemorrhagic lesions was EHDV-2 isolated and/or erythrocyte-associated EHDV-2 RNA detected by serotype-specific reverse transcription (RT)-PCR. By using a novel RT in situ PCR assay, viral nucleic acid was localized to the cytoplasm of large numbers of tissue leukocytes and vascular endothelium in tissues with hemorrhage and to vessels, demonstrating acute intimal and medial necrosis. Because PCR amplification prior to in situ hybridization was essential for detecting EHDV, the virus copy number within individual cells was low, <20 virus copies. These findings suggest that massive covert infection characterized by rapid dissemination of virus facilitates the severe and lethal nature of this disease.
Transactivation-Competent Bovine Papillomavirus E2 Protein Is Specifically Required for Efficient Repression of Human Papillomavirus Oncogene Expression and for Acute Growth Inhibition of Cervical Carcinoma Cell Lines
Authors: Goodwin Edward C. , Naeger Lisa Kay , Breiding David E. , Androphy Elliot J. , DiMaio Daniel 
Published: December 2019

ABSTRACT

The papillomavirus E2 proteins can function as sequence-specific transactivators or transrepressors of transcription and as cofactors in viral DNA replication. We previously demonstrated that acute expression of the bovine papillomavirus type 1 (BPV1) E2 protein in HeLa and HT-3 cervical carcinoma cell lines greatly reduced cellular proliferation by imposing a specific G
1
/S phase growth arrest. In this report, we analyzed the effects of a panel of point mutations in the BPV1 E2 protein to identify the functional requirements for acute growth inhibition. Disruption of E2-specific transactivation by mutations within either the transactivation domain or the DNA binding domain severely impaired E2-mediated growth inhibition in HeLa and HT-3 cells, even though these mutants retain various other E2 activities. This result indicates that functional transactivation activity is required for acute E2-mediated growth inhibition. HeLa cells, which contain a wild-type p53 gene, and HT-3 cells, which contain a transactivation-defective p53 gene, exhibited similar responses to the E2 mutants, suggesting that identical functions of the E2 protein were required for growth arrest regardless of p53 status. Replacement of the E2 transactivation domain with that of the herpes simplex virus VP16 generated a chimeric transactivator that efficiently stimulated expression of an E2-responsive reporter plasmid yet was completely defective for growth inhibition, suggesting that an E2-specific transactivation function is required for growth arrest. Surprisingly, the transactivation-defective E2 mutants were also markedly defective in their ability to repress transcription of the native human papillomavirus type 18 (HPV18) E6/E7 oncogenes in HeLa cells and of the HPV18 promoter present in a transfected reporter plasmid. These mutants were also defective in their ability to increase p53 levels. Therefore, efficient repression of the HPV18 promoter in HeLa cells is not merely a consequence of the binding of an E2 protein to appropriately situated binding sites in the promoter.
The Epstein-Barr Virus BARF1 Gene Encodes a Novel, Soluble Colony-Stimulating Factor-1 Receptor
Authors: Strockbine Laura D. , Cohen Jeffrey I. , Farrah Terry , Lyman Stewart D. , Wagener Felecia , DuBose Robert F. , Armitage Richard J. , Spriggs Melanie K. 
Published: December 2019

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with infectious mononucleosis and several tumors. The BARF1 gene is transcribed early after EBV infection from theBamHI A fragment of the EBV genome. Evidence shown here indicates that the BARF1 protein is secreted into the medium of transfected cells and from EBV-carrying B cells induced to allow lytic replication of the virus. Expression cloning identified colony-stimulating factor-1 (CSF-1) as a ligand for BARF1. Computer-assisted analyses indicated that subtle amino acid sequence homology exists between BARF1 and c-fms, the cellular proto-oncogene that is the receptor for CSF-1. Recombinant BARF1 protein was found to be biologically active, and it neutralized the proliferative effects of human CSF-1 in a dose-dependent fashion when assayed in vitro. Since CSF-1 is a pleiotropic cytokine best known for its differentiating effects on macrophages, these data suggest that BARF1 may function to modulate the host immune response to EBV infection.
The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes
Authors: Johnson Rowena J. , Stack Maria , Hazlewood Sheila A. , Jones Matthew , Blackmore Colin G. , Hu Li-Fu , Rowe Martin 
Published: December 2019

ABSTRACTOne group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.
A Tandem Array of Minimal U1 Small Nuclear RNA Genes Is Sufficient To Generate a New Adenovirus Type 12Inducible Chromosome Fragile Site
Authors: Li Zengji , Bailey Arnold D. , Buchowski Jacob , Weiner Alan M. 
Published: December 2019

ABSTRACT

Infection of human cells with adenovirus serotype 12 (Ad12) induces metaphase fragility of four, and apparently only four, chromosomal loci. Surprisingly, each of these four loci corresponds to a cluster of genes encoding a small abundant structural RNA: the
RNU1
and
RNU2
loci contain tandemly repeated genes encoding U1 and U2 small nuclear RNAs (snRNAs), respectively; the
PSU1
locus is a cluster of degenerate U1 genes; and the
RN5S
locus contains the tandemly repeated genes encoding 5S rRNA. These observations suggested that high local levels of transcription, in combination with Ad12 early functions, can interfere with metaphase chromatin packing. In support of this hypothesis, we and others found that an artificial tandem array of transcriptionally active, but not inactive, U2 snRNA genes would generate a novel Ad12-inducible fragile site. Although U1 and U2 snRNA are both transcribed by RNA polymerase II and share similar enhancer, promoter, and terminator signals, the human U1 promoter is clearly more complex than that of U2. In addition, the natural U1 tandem repeat unit exceeds 45 kb, whereas the U2 tandem repeat unit is only 6.1 kb. We therefore asked whether an artificial array of minimal U1 genes would also generate a novel Ad12-inducible fragile site. The exogenous U1 genes were marked by an innocuous U72C point mutation within the U1 coding region so that steady-state levels of U1 snRNA derived from the artificial array could be quantified by a simple primer extension assay. We found that the minimal U1 genes were efficiently expressed and were as effective as minimal U2 genes in generating a novel Ad12-inducible fragile site. Thus, despite significant differences in promoter architecture and overall gene organization, the active U1 transcription units suffice to generate a new virally inducible fragile site.
A Role for the Sendai Virus P Protein Trimer in RNA Synthesis
Authors: Curran Joseph 
Published: December 2019

ABSTRACTThe SeV P protein is found as a homotrimer (P3) when it is expressed in mammalian cells, and trimerization is mediated by a predicted coiled-coil motif which maps within amino acids (aa) 344 to 411 (the BoxA region). The bacterially expressed protein also appears to be trimeric, apparently precluding a role for phosphorylation in the association of the P monomers. I have examined the role of P trimerization both in the protein’s interaction with the nucleocapsid (N:RNA) template and in the protein’s function on the template during RNA synthesis. As with the results of earlier experiments (32), I found that both the BoxA and BoxC (aa 479 to 568) regions were required for stable binding of P to the N:RNA. Binding was also observed with P proteins containing less than three BoxC regions, suggesting that trimerization may be required to permit contacts between multiple BoxC regions and the N:RNA. However, these heterologous trimers failed to function in viral RNA synthesis, indicating that the third C-terminal leg of the trimer plays an essential role in P function on the template. We speculate that this function may involve the movement of P (and possibly the polymerase complex) on the template and the maintenance of processivity.
gag
,
vif
, and
nef
Genes Contribute to the Homologous Viral Interference Induced by a Nonproducer Human Immunodeficiency Virus Type 1 (HIV-1) Variant: Identification of Novel HIV-1-Inhibiting Viral Protein Mutants
Authors: D’Aloja Paola , Olivetta Eleonora , Bona Roberta , Nappi Filomena , Pedacchia Daniela , Pugliese Katherina , Ferrari Giuliana , Verani Paola , Federico Maurizio 
Published: December 2019

ABSTRACT

We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV
gag
,
vif
, or
nef
gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the
tat
,
rev
, or
vpu
F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV
vpr
,
pol
, or
env
gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV
vif
or
nef
allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both
vif
-permissive and
vif
-nonpermissive cells. The levels of action of F12-HIV
vif
and
nef
anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV
vif
-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy.
Virus-Specific CD4+T Cells Eliminate Borna Disease Virus from the Brain via Induction of Cytotoxic CD8+T Cells
Authors: Nöske Kerstin , Bilzer Thomas , Planz Oliver , Stitz Lothar 
Published: December 2019

ABSTRACTPersistent Borna disease virus infection of the brain can be prevented by treatment of naive rats with a virus-specific CD4+T-cell line prior to infection. In rats receiving this treatment, only a transient low-level encephalitis was seen compared to an increasingly inflammatory reaction in untreated infected control rats. Virus replication was found in the brain for several days after infection before the virus was cleared from the central nervous system. The loss of infectivity from the brain was confirmed by negative results by reverse transcription-PCR with primers for mRNA, by in situ hybridization for both genomic and mRNA, and by immunohistology. Most importantly, in vitro assays revealed that the T-cell line used for transfusion had no cytotoxic capacity. The kinetics of virus clearance were paralleled by the appearance of CD8+T cells and the expression of perforin in the brain. Testing of lymphocytes isolated from the brains of CD4+T-cell-treated rats after challenge revealed high cytotoxic activity due to the presence of CD8+cytotoxic T cells at time points when brain lymphocytes from infected control rats induced low-level cytolysis of target cells. Neutralizing antiviral antibodies and gamma interferon were shown not to be involved in the elimination of virus from the brain.
Human T-Cell Leukemia Virus Type 1 pX-I and pX-II Open Reading Frames Are Dispensable for the Immortalization of Primary Lymphocytes
Authors: Robek Michael D. , Wong Fen-Hwa , Ratner Lee 
Published: December 2019

ABSTRACT

Human T-cell leukemia virus type 1 (HTLV-1) infects and transforms CD4
+
T-lymphocytes both in vivo and in vitro. Although the Tax protein of HTLV-1 has been strongly implicated as a transforming agent, other virally encoded proteins may also play a role in the transformation process. In addition to the
rex
and
tax
genes, the pX region of the HTLV-1 genome contains two open reading frames (pX-I and pX-II) which encode the putative viral accessory proteins known as p12
I
, p30
II
, and p13
II
. Mutations in the ACH molecular clone of HTLV-1 that are predicted to abrogate the expression of p12
I
, p13
II
and p30
II
were constructed. These mutations had no effect on viral replication or the immortalization of primary lymphocytes. Although these proteins are dispensable for viral replication and immortalization in vitro, it remains possible that they alter infection in vivo.
Recombinant Respiratory Syncytial Virus (RSV) Bearing a Set of Mutations from Cold-Passaged RSV Is Attenuated in Chimpanzees
Authors: Whitehead Stephen S. , Juhasz Katalin , Firestone Cai-Yen , Collins Peter L. , Murphy Brian R. 
Published: December 2019

ABSTRACT

A set of five missense mutations previously identified by nucleotide sequence analysis of subgroup A cold-passaged (
cp
) respiratory syncytial virus (RSV) has been introduced into a recombinant wild-type strain of RSV. This recombinant virus, designated rA2cp, appears to replicate less efficiently in the upper and lower respiratory tracts of seronegative chimpanzees than either biologically derived or recombinant wild-type RSV. Infection with rA2cp also resulted in significantly less rhinorrhea and cough than infection with wild-type RSV. These findings confirm the role of the
cp
mutations in attenuation of RSV and identify their usefulness for inclusion in future live attenuated recombinant RSV vaccine candidates.