bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rabbit Genital Tissue Is Susceptible to Infection by Rabbit Oral Papillomavirus: an Animal Model for a Genital Tissue-Targeting Papillomavirus
Authors: Harvey Stephen B. , Cladel Nancy M. , Budgeon Lynn R. , Welsh Patricia A. , Griffith James W. , Lang C. Max , Christensen Neil D. 
Published: December 2019

ABSTRACT
Rabbit oral papillomavirus (ROPV) is a mucosatropic papillomavirus which naturally infects oral mucosal sites of domestic rabbits. In this study, we tested the hypothesis that rabbit genital mucosa is also susceptible to ROPV infection by using the athymic mouse xenograft system and adult immunocompetent rabbits. Subrenal xenografts of ROPV-infected rabbit vulvar and penile sheath tissues were strongly positive for ROPV infection by histologic, in situ hybridization, and Southern analyses. Direct inoculation of adult rabbit penises with infectious ROPV produced small raised lesions of approximately 1 by 1 by 1 mm that were ROPV positive by both in situ hybridization and Southern analyses and were also viral capsid antigen positive by immunohistological staining. Infection of rabbit genital tissues with ROPV may be a useful animal model for the study of genital tissue-targeting papillomaviruses.
Recombination of Engineered Defective RNA Species Produces Infective Potyvirus In Planta
Authors: Gal-On Amit , Meiri Eti , Raccah Benjamin , Gaba Victor 
Published: December 2019

ABSTRACT
Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time.
Identification of Replication-Competent Strains of Simian Immunodeficiency Virus Lacking Multiple Attachment Sites for N-Linked Carbohydrates in Variable Regions 1 and 2 of the Surface Envelope Protein
Authors: Reitter Julie N. , Desrosiers Ronald C. 
Published: December 2019

ABSTRACT
Carbohydrates comprise about 50% of the mass of gp120, the external envelope glycoprotein of simian immunodeficiency virus (SIV) and human immunodeficiency virus. We identified 11 replication-competent derivatives of SIVmac239 lacking two, three, four, or five potential sites for N-linked glycosylation. These sites were located within and around variable regions 1 and 2 of the surface envelope protein of the virus. Asn (AAT) of the canonical N-linked glycosylation recognition sequence (Asn X Ser/Thr) was changed in each case to the structurally similar Gln (CAG or CAA) such that two nucleotide changes in the codon would be required for reversion. Replication of one triple mutant (g456), however, was severely impaired. A revertant of the g456 mutant was recovered from CEMx174 cells with a Met-to-Val compensatory substitution at position 144, 2 amino acids upstream of attachment site 5. Thus, a debilitating loss of sites for N-linked glycosylation can be compensated for by amino acid changes not involving the Asn-X-Ser/Thr consensus motif. These results provide a framework to begin testing the hypothesis that carbohydrates form a barrier that can limit the humoral immune responses to the virus.
Preferential Completion of Human Immunodeficiency Virus Type 1 Proviruses Initiated with tRNA
3
Lys
rather than tRNA
1,2
Lys
Authors: Zhang Zhijun , Yu Qin , Kang Sang-Moo , Buescher James , Morrow Casey D. 
Published: December 2019

ABSTRACT

All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA
3
Lys
. However, within HIV-1 virions, there are approximately equal amounts of tRNA
1
Lys
, tRNA
2
Lys
, and tRNA
3
Lys
. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA
3
Lys
, tRNA
1
Lys
and tRNA
2
Lys
could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a
gpt
gene encoding xanthine-guanine phosphoribosyl transferase in place of the
env
gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA
3
Lys
, not tRNA
1
Lys
or tRNA
2
Lys
. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA
1,2
Lys
. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA
3
Lys
from proviral DNA isolated from
gpt
-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA
1,2
Lys
resulted in
gpt
- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA
1,2
Lys
. The results of these studies suggest that the selection of tRNA
3
Lys
for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.
Intracellular Signaling by the Chemokine Receptor US28 during Human Cytomegalovirus Infection
Authors: Billstrom Marcella A. , Johnson Gary L. , Avdi Natalie J. , Worthen G. Scott 
Published: December 2019

ABSTRACTIn patients with impaired cell-mediated immune responses (e.g., lung transplant recipients and AIDS patients), cytomegalovirus (CMV) infection causes severe disease such as pneumonitis. However, although immunocompetency in the host can protect from CMV disease, the virus persists by evading the host immune defenses. A model of CMV infection of the endothelium has been developed in which inflammatory stimuli, such as the CC chemokine RANTES, bind to the endothelial cell surface, stimulating calcium flux during late times of CMV infection. At 96 h postinfection, CMV-infected cells express mRNA of the CMV-encoded CC chemokine receptor US28 but do not express mRNA of other CC chemokine receptors that bind RANTES (CCR1, CCR4, CCR5). Cloning and stable expression of the receptor CMV US28 in human kidney epithelial cells (293 cells) with and without the heterotrimeric G protein α16indicated that CMV US28 couples to both Gαiand Gα16proteins to activate calcium flux in response to the chemokines RANTES and MCP-3. Furthermore, cells that coexpress US28 and Gα16responded to RANTES stimulation with activation of extracellular signal-regulated kinase, which could be attributed, in part, to specific Gα16coupling. Thus, through expression of the CC chemokine receptor US28, CMV may utilize resident G proteins of the infected cell to manipulate cellular responses stimulated by chemokines.
Role of the Pre-S2 Domain of the Large Envelope Protein in Hepatitis B Virus Assembly and Infectivity
Authors: Le Seyec J. , Chouteau P. , Cannie I. , Guguen-Guillouzo C. , Gripon P. 
Published: December 2019

ABSTRACT
Among the three viral proteins present in the hepatitis B virus (HBV) envelope, both the small and large polypeptides, but not the middle polypeptide, are necessary for the production of complete viral particles. Whereas it has been established that the C-terminal extremity of the pre-S1 region is required for HBV morphogenesis, whether the pre-S2 region of the large surface protein plays a critical role remains questionable. In the present study, we have analyzed the role of the large-polypeptide pre-S2 region in viral maturation and infectivity. For this purpose, mutants bearing contiguous deletions covering the entire pre-S2 domain were generated. First, the efficient expression of all the mutant large envelope proteins was verified and their ability to substitute for the wild-type form in virion secretion was tested. We found that distinct deletions covering the domain between amino acids 114 and 163 still allowed virion production. In contrast, the polypeptide lacking the first 5 amino acids of pre-S2 (amino acids 109 to 113) was unable to support viral secretion. This result shows that the domain of the large surface protein, required for this process, must be extended to the N-terminal extremity of pre-S2. We then demonstrated that all the mutants competent for virion release were able to infect normal human hepatocytes in primary culture. Taken together, these results indicate that only 10% of the large-protein pre-S2 region at its N-terminal extremity is essential for virion export and that the remaining part, dispensable for viral secretion, is also dispensable for infectivity.
Particle Polymorphism Caused by Deletion of a Peptide Molecular Switch in a Quasiequivalent Icosahedral Virus
Authors: Dong X. Fan , Natarajan Padmaja , Tihova Mariana , Johnson John E. , Schneemann Anette 
Published: December 2019

ABSTRACT
The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.
Structure and Phylogenetic Analysis of an Endogenous Retrovirus Inserted into the Human Growth Factor Gene Pleiotrophin
Authors: Schulte Anke M. , Wellstein Anton 
Published: December 2019

ABSTRACTA human endogenous retrovirus-like element (HERV), flanked by long terminal repeats of 502 and 495 nucleotides is inserted into the human pleiotrophin (PTN) gene upstream of the open reading frame. Based on its Glu-tRNA primer binding site specificity and the location within the PTN gene, we named this element HERV-E.PTN. HERV-E.PTN appears to be a recombined viral element based on its high homology (70 to 86%) in distinct areas to members of two distantly related HERV type C families, HERV-E and retrovirus-like element I (RTVL-I). Furthermore, its pseudogene region is organized from 5′ to 3′ intogag-,pol-,env-,pol-,env-similar sequences. Interestingly, full-length and partial HERV-E.PTN-homologous sequences were found in the human X chromosome, the human hereditary haemochromatosis region, and the BRCA1 pseudogene. Finally, Southern analyses indicate that the HERV-E.PTN element is present in the PTN gene of humans, chimpanzees, and gorillas but not of rhesus monkeys, suggesting that genomic insertion occurred after the separation of monkeys and apes about 25 million years ago.
Heparan Sulfate Proteoglycan Binding by Herpes Simplex Virus Type 1 Glycoproteins B and C, Which Differ in Their Contributions to Virus Attachment, Penetration, and Cell-to-Cell Spread
Authors: Laquerre Sylvie , Argnani Rafaela , Anderson Dina B. , Zucchini Silvia , Manservigi Roberto , Glorioso Joseph C. 
Published: December 2019

ABSTRACT

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB’s role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK
−
also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK
−
gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK
−
. Compared with wild-type virus, KgBpK
−
showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the
lacZ
gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK
−
gC
−
) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK
−
mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.
Role of E4 in Eliciting CD4 T-Cell and B-Cell Responses to Adenovirus Vectors Delivered to Murine and Nonhuman Primate Lungs
Authors: Chirmule Narendra , Hughes Joseph V. , Gao Guang-Ping , Raper Steven E. , Wilson James M. 
Published: December 2019

ABSTRACT
Adenovirus vectors delivered to lung are being considered in the treatment of cystic fibrosis (CF). Vectors from which E1 has been deleted elicit T- and B-cell responses which confound their use in the treatment of chronic diseases such as CF. In this study, we directly compare the biology of an adenovirus vector from which E1 has been deleted to that of one from which E1 and E4 have been deleted, following intratracheal instillation into mouse and nonhuman primate lung. Evaluation of the E1 deletion vector in C57BL/6 mice demonstrated dose-dependent activation of both CD4 T cells (i.e., TH1 and TH2 subsets) and neutralizing antibodies to viral capsid proteins. Deletion of E4 and E1 had little impact on the CD4 T-cell proliferative response and cytolytic activity of CD8 T cells against target cells expressing viral antigens. Analysis of T-cell subsets from mice exposed to the vector from which E1 and E4 had been deleted demonstrated preservation of TH1 responses with markedly diminished TH2 responses compared to the vector with the deletion of E1. This effect was associated with reduced TH2-dependent immunoglobulin isotypes and markedly diminished neutralizing antibodies. Similar results were obtained in nonhuman primates. These studies indicate that the vector genotype can modify B-cell responses by differential activation of TH1 subsets. Diminished humoral immunity, as was observed with the E1 and E4 deletion vectors in lung, is indeed desired in applications of gene therapy where readministration of the vector is necessary.
Inhibition of Simian Immunodeficiency Virus (SIV) Replication by CD8+T Lymphocytes from Macaques Immunized with Live Attenuated SIV
Authors: Gauduin Marie-Claire , Glickman Rhona L. , Means Robert , Johnson R. Paul 
Published: December 2019

ABSTRACTCharacterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8+T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Δnef or SIVmac239Δ3 to inhibit SIV replication. CD8+T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4+T cells. Suppression of SIV replication by unstimulated CD8+T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8+T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8+T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8+cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1α (MIP-1α), or MIP-1β from stimulated CD8+T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8+T cells of control animals. However, addition of antibodies that neutralize these β-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8+T cells. Our results indicate that inhibition of SIV replication by CD8+T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1α, and MIP-1β.
Viral Genetic Evolution in Macaques Infected with Molecularly Cloned Simian Immunodeficiency Virus Correlates with the Extent of Persistent Viremia
Authors: Hirsch Vanessa M. , Dapolito George , Hahn Anna , Lifson Jeff , Montefiori David , Brown Charles R. , Goeken Robert 
Published: December 2019

ABSTRACTGenetic evolution of the simian immunodeficiency virus (SIV) envelope glycoprotein was evaluated in a group of six macaques (Macaca nemestrina) infected with the molecularly cloned, moderately pathogenic SIVsm62d. The extent of envelope evolution was subsequently evaluated within the context of the individual pattern of viremia and disease outcome. Two macaques in this cohort developed AIDS by 1.5 years postinoculation (progressors), whereas the remaining four macaques remained asymptomatic (nonprogressors). Compared with the nonprogressor macaques, the two progressor macaques exhibited higher persistent plasma viremia, higher homologous neutralizing antibody titers, and more extensive mutation and evolution in the V1 region of envelope. Although clearly distinct in each of these parameters from the progressors, the four nonprogressors exhibited more individual variability with respect to the extent of persistent viremia and genetic evolution of the V1 region of envelope. The extent of V1 envelope varied from no apparent V1 evolution in a macaque with good viral containment to extensive evolution in one macaque with persistent viremia. This study underscores the critical role of persistent replication in the genetic evolution of SIV.
A Role for Herpesvirus Saimiri orf14 in Transformation and Persistent Infection
Authors: Duboise Monroe , Guo Jie , Czajak Sue , Lee Heuiran , Veazey Ronald , Desrosiers Ronald C. , Jung Jae U. 
Published: December 2019

ABSTRACTThe product of open reading frame 14 (orf14) of herpesvirus saimiri (HVS) exhibits significant homology with mouse mammary tumor virus superantigen. orf14 encodes a 50-kDa secreted glycoprotein, as shown previously (Z. Yao, E. Maraskovsky, M. K. Spriggs, J. I. Cohen, R. J. Armitage, and M. R. Alderson, J. Immunol. 156:3260–3266, 1996). orf14 expressed from recombinant baculovirus powerfully induces proliferation of CD4-positive cells originating from several different species. To study the role of orf14 in transformation, a mutant form of HVS (HVS Δorf14) was constructed with a deletion in the orf14 gene. The transforming potential of HVS Δorf14 was tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, while the HVS Δorf14 mutant did not produce such a growth transformation. In addition, HVS Δorf14 was nononcogenic in common marmosets. In contrast to other nononcogenic HVS mutant viruses which were repeatedly isolated from peripheral blood mononuclear cells of infected marmosets for more than 5 months, HVS Δorf14 did not persist at a high level in vivo. These results demonstrate that orf14 of HVS is not required for replication but is required for transformation and for high-level persistence in vivo.
Localization of Varicella-Zoster Virus Gene 21 Protein in Virus-Infected Cells in Culture
Authors: Mahalingam Ravi , Lasher Robert , Wellish Mary , Cohrs Randall J. , Gilden Donald H. 
Published: December 2019

ABSTRACT

Although four varicella-zoster virus (VZV) genes have been shown to be transcribed in latently infected human ganglia, their role in the development and maintenance of latency is unknown. To study these VZV transcripts, we decided first to localize their expression products in productively infected cells. We began with VZV gene 21, whose open reading frame (ORF) is 3,113 bp. We cloned the 5′ and 3′ ends and the predicted antigenic segments of the ORF as 1292-, 1280-, and 880-bp DNA fragments, respectively, into the prokaryotic expression vector pGEX-2T. The three VZV 21 ORFs were expressed as approximately 75-, 73-, and 59-kDa glutathione
S
-transferase fusion proteins in
Escherichia coli
. To prepare polyclonal antibodies that would recognize all potential epitopes on the VZV gene 21 protein, rabbits were inoculated with a mixture of the three fusion proteins, and antisera were obtained and affinity purified. Immunohistochemical and immunoelectron microscopic analyses using these antibodies revealed VZV ORF 21 protein in both the nucleus and cytoplasm of VZV-infected cells. When these antibodies were applied to purified VZV nucleocapsids, intense staining was seen in their central cores.
Circumvention of Immunity to the Adenovirus Major Coat Protein Hexon
Authors: Roy Soumitra , Shirley Pamela S. , McClelland Alan , Kaleko Michael 
Published: December 2019

ABSTRACT
Immunity to adenoviruses is an important hurdle to be overcome for successful gene therapy. The presence of antibodies to the capsid proteins prevents efficacious adenovirus vector administration in vivo. We tested whether immunity to a particular serotype of adenovirus (Ad5) may be overcome with a vector that encodes the hexon sequences from a different adenovirus serotype (Ad12). We successfully constructed an adenovirus vector with a chimeric Ad5-Ad12 hexon which was not neutralized by plasma from C57BL/6 mice immunized with Ad5. The vector was also capable of transducing the livers of C57BL/6 mice previously immunized with Ad5.