bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunity in Chimpanzees Chronically Infected with Hepatitis C Virus: Role of Minor Quasispecies in Reinfection
Authors: Wyatt Colby A. , Andrus Linda , Brotman Betsy , Huang Fannie , Lee Dong-Hun , Prince Alfred M. 
Published: December 2019

ABSTRACT
We have previously reported that chimpanzees chronically infected with hepatitis C virus (HCV) could be reinfected, even with the original infecting strain. In this study we tested the hypothesis that this might reflect the presence of minor quasispecies to which there was little or no immunity. To evaluate this hypothesis, we sequenced multiple clones taken at intervals after primary infection and rechallenge from four chronically infected chimpanzees. The inoculum used in these studies (HCV-H, genotype 1a) revealed 17 separate variants among 46 clones sequenced. Following challenge, each of the four challenged animals showed marked alterations of their quasispecies distribution. The new variants, which appeared 1 to 6 weeks after challenge, were either identical to or closely resembled variants present in the challenge inoculum. These results, paralleled by an increase in viremia in some of the challenged animals, suggest that quasispecies in the challenge inoculum were responsible for signs of reinfection and that there was little immunity. However, the newly emerged quasispecies completely took over infection in only one animal. In the remaining three chimpanzees the prechallenge quasispecies were able to persist. The natural evolution of infection within chimpanzees resulted in variants able to compete with the inoculum variants. Whether through reexposure or the natural progression of infection, newly emerged quasispecies are likely to play a role in the pathogenesis of chronic HCV infection.
Preemptive CD8 T-Cell Immunotherapy of Acute Cytomegalovirus Infection Prevents Lethal Disease, Limits the Burden of Latent Viral Genomes, and Reduces the Risk of Virus Recurrence
Authors: Steffens Hans-Peter , Kurz Sabine , Holtappels Rafaela , Reddehase Matthias J. 
Published: December 2019

ABSTRACT
In the immunocompetent host, primary cytomegalovirus (CMV) infection is resolved by the immune response without causing overt disease. The viral genome, however, is not cleared but is maintained in a latent state that entails a risk of virus recurrence and consequent organ disease. By using murine CMV as a model, we have shown previously that multiple organs harbor latent CMV and that reactivation occurs with an incidence that is determined by the viral DNA load in the respective organ (M. J. Reddehase, M. Balthesen, M. Rapp, S. Jonjic, I. Pavic, and U. H. Koszinowski. J. Exp. Med. 179:185–193, 1994). This predicts that a therapeutic intervention capable of limiting the load of latent viral genome should also reduce the risk of virus recurrence. Here we demonstrate the benefits and the limits of a preemptive CD8 T-cell immunotherapy of CMV infection in the immunocompromised bone marrow transplantation recipient. Antiviral CD8 T cells prevented CMV disease and accelerated the resolution of productive infection. The therapy also resulted in a lower load of latent CMV DNA in organs and consequently reduced the incidence of recurrence. The data thus provide a further supporting argument for clinical trials of preemptive cytoimmunotherapy of human CMV disease with CD8 T cells. However, CD8 T cells failed to clear the viral DNA. The therapy-susceptible portion of the DNA load differed between organs and was highest in the lungs. The existence of an invariant, therapy-resistant load suggests a role for immune system evasion mechanisms in the establishment of CMV latency.
Identification of Positive and Negative Regulatory Regions Involved in Regulating Expression of the Human Cytomegalovirus UL94 Late Promoter: Role of IE2-86 and Cellular p53 in Mediating Negative Regulatory Function
Authors: Wing Bret A. , Johnson Robert A. , Huang Eng-Shang 
Published: December 2019

ABSTRACT

The human cytomegalovirus (HCMV) UL94 gene product is a herpesvirus-common virion protein that is expressed with true late kinetics. To identify the important
cis
- and
trans
-acting factors which contribute to UL94 transcriptional regulation, we have cloned, sequenced, and analyzed UL94 promoter function by transient transfection analysis. Transfection of UL94 promoter-reporter gene constructs into permissive human fibroblasts or U373(MG) cells indicated that promoter activity was detected following infection with HCMV. Point mutations within a TATA-like element located upstream of the RNA start site significantly reduced UL94 promoter activity. Deletion mutagenesis of the promoter indicated that a positive regulatory element (PRE) was likely to exist downstream of the UL94 mRNA start site, while a negative regulatory element (NRE) was present upstream of the TATA box. At late times of infection, the PRE appeared to have a dominant effect over the NRE to stimulate maximum levels of UL94 promoter activity, while at earlier times of infection, no activity associated with the PRE could be detected. The NRE, however, appeared to cause constitutive down-regulation of UL94 promoter activity. Binding sites for the cellular p53 protein located within the NRE appeared to contribute to NRE function, and NRE function could be recapitulated in cotransfection assays by concomitant expression of p53 and HCMV IE2-86 protein. Our results suggest a novel mechanism by which the cellular protein p53, which is involved in both transcriptional regulation and progression of cellular DNA synthesis, plays a central role in the regulation of a viral promoter which is not activated prior the onset of viral DNA replication.
Human Endogenous Retrovirus K Homologous Sequences and Their Coding Capacity in Old World Primates
Authors: Mayer Jens , Meese Eckart , Mueller-Lantzsch Nikolaus 
Published: December 2019

ABSTRACT

The coding capacity for retroviral Gag and Env proteins has been maintained in human endogenous retroviruses of the HERV-K family. HERV-K homologous sequences have been found in all Old World primates. Here, we examined Old World primate species for the presence of full-length HERV-K
gag
and
env
genes and the presence of
gag
and
env
open reading frames as determined by the protein truncation test. Full-length HERV-K
env
genes were found in DNAs of all Old World primate species, whereas open reading frames for Env protein were found solely in human, chimpanzee, and gorilla DNAs. The mutational event leading to two HERV-K types was found to have occurred after the separation of hominids from lower Old World primates and before the expansion of hominids. Full-length HERV-K
gag
genes in hominids displayed a 96-bp deletion compared to those in lower Old World primates. The ancient
gag
variant has not been maintained during hominid evolution. Open reading frames for HERV-K Gag have been found in all Old World primates except chimpanzees. Our study of the HERV-K family during Old World primate evolution contributes to the understanding of their possible biological functions in the host genomes.
Competition for DNA Binding Sites between the Short and Long Forms of E2 Dimers Underlies Repression in Bovine Papillomavirus Type 1 DNA Replication Control
Authors: Lim Daniel A. , Gossen Manfred , Lehman Chris W. , Botchan Michael R. 
Published: December 2019

ABSTRACT
Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.
Pseudorabies Virus Glycoprotein gK Is a Virion Structural Component Involved in Virus Release but Is Not Required for Entry
Authors: Klupp Barbara G. , Baumeister Judith , Dietz Petra , Granzow Harald , Mettenleiter Thomas C. 
Published: December 2019

ABSTRACT

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione
S
-transferase–gK fusion protein expressed in
Escherichia coli
. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with
N
-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-
lacZ
expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB
−
genome (PrV-gK
gB
). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.
The Open Reading Frame (ORF) 50a Gene Product Regulates ORF 57 Gene Expression in Herpesvirus Saimiri
Authors: Whitehouse Adrian , Cooper Matthew , Hall Kersten T. , Meredith David M. 
Published: December 2019

ABSTRACTWe have previously demonstrated that open reading frame (ORF) 50 and ORF 57 encode transcriptional regulating genes in herpesvirus saimiri. ORF 50, a homolog of Epstein-Barr virus R protein, is a sequence-specific transactivator, whereas ORF 57 acts posttranscriptionally. In this report, we demonstrate that the ORF 57 gene is regulated by the ORF 50a gene product. We show that the ORF 57 gene is expressed at basal levels early in the virus replication cycle and that thereafter it is transactivated by the ORF 50a gene product, due to an increase in RNA levels. As it has been shown that the ORF 57 gene product downregulates ORF 50a due to the presence of its intron, these combined observations identify a feedback mechanism modulating gene expression in herpesvirus saimiri, whereby ORF 50a transcription is downregulated by the ORF 57 gene product, a gene which it specifically transactivates. Furthermore, we propose that the intron-containing ORF 57 gene downregulates itself by the same mechanism as that for ORF 50a, as both genes are downregulated at similar times during the replication cycle.
Association of the Human Papillomavirus Type 11 E1 Protein with Histone H1
Authors: Swindle C. Scott , Engler Jeffrey A. 
Published: December 2019

ABSTRACT
The E1 and E2 proteins are the only virus-encoded factors required for human papillomavirus (HPV) DNA replication. The E1 protein is a DNA helicase responsible for initiation of DNA replication at the viral origin. Its recruitment to the origin is facilitated by binding to E2, for which specific recognition elements are located at the origin. The remaining replication functions for the virus, provided by the host cell’s replication machinery, may be mediated by further interactions with E1 and E2. Histone H1 was identified as an HPV type 11 (HPV-11) E1-binding protein by far-Western blotting and by microsequence analyses of a 34-kDa protein purified by E1 affinity chromatography. E1 also bound in vitro to H1 isolated under native conditions in association with intact nucleosomes. In addition, E1 and H1 were coimmunoprecipitated by an E1 antiserum from a nuclear extract prepared from cells expressing recombinant E1. Bound H1 was displaced from HPV-11 DNA by the addition of E1, suggesting that E1 can promote replication initiation and elongation by alteration of viral chromatin structure and disruption of nucleosomes at the replication fork. Furthermore, a region of the HPV-11 genome containing the origin of replication was identified which had weaker affinity for H1 than that of the remaining genome. This result suggests that the presence of a DNA structure at or near the HPV origin facilitates initiation of DNA replication by exclusion of H1. These results are similar to those of studies of simian virus 40 DNA replication, in which a large T antigen-H1 interaction and an H1-resistant region at the origin of DNA replication have also been demonstrated.
Modulation of Activity of Moloney Murine Leukemia Virus Preintegration Complexes by Host Factors In Vitro
Authors: Li Ling , Farnet Chris M. , Anderson W. French , Bushman Frederic D. 
Published: December 2019

ABSTRACT
We have explored the requirements for host proteins in the integration of Moloney murine leukemia virus (MoMuLV) cDNA in vitro. Following infection, it is possible to lyse cells and obtain preintegration complexes (PICs) capable of integrating the MoMuLV cDNA into an added target DNA in vitro (intermolecular integration). PICs can be stripped of required proteins by gel filtration in high-salt buffers (600 mM KCl), allowing the nature of the removed factors to be investigated by in vitro reconstitution. In a previous study of human immunodeficiency virus type 1 (HIV-1) PICs, the host protein HMG I(Y) was found to be able to restore activity to salt-stripped PICs. In contrast, salt stripping and reconstitution of MoMuLV PICs led to the proposal that a host factor is important for a different activity, blocking integration into the cDNA itself (autointegration). In this report, we investigated reconstitution of salt-stripped MoMuLV PICs and found that addition of cellular extract from uninfected NIH 3T3 cells could block autointegration and also restore intermolecular integration. Isolation of the intermolecular integration-complementing activity yielded HMG I(Y), as in the HIV-1 case. However, HMG I(Y) could not block autointegration, implicating a different host factor in this process. Additionally, when MoMuLV PICs were partially purified but not salt stripped, the intermolecular integration activity was reduced but could be stimulated by the addition of any of several purified DNA binding proteins. In summary, three activities were detected: (i) the intermolecular integration cofactor HMG I(Y), (ii) an autointegration barrier protein, and (iii) stimulatory DNA binding proteins.
Signal Peptidase Cleavage at the Flavivirus C-prM Junction: Dependence on the Viral NS2B-3 Protease for Efficient Processing Requires Determinants in C, the Signal Peptide, and prM
Authors: Stocks C. E. , Lobigs M. 
Published: December 2019

ABSTRACTSignal peptidase cleavage at the C-prM junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the COOH terminus of the C protein. The signal peptidase cleavage occurs efficiently in circumstances where the C protein is deleted or if the viral protease complex is present. In this study, we used cDNA of Murray Valley encephalitis virus (MVE) to examine features of the structural polyprotein which allow this regulation of a luminal cleavage by a cytoplasmic protease. We found that the inefficiency of signal peptidase cleavage in the absence of the viral protease is not attributable solely to features of the C protein. Inhibition of cleavage still occurred when charged residues in C were mutated to uncharged residues or when an unrelated protein sequence (that of ubiquitin) was substituted for C. Also, fusion of the C protein did not inhibit processing of an alternative adjacent signal sequence. The cleavage region of the flavivirus prM translocation signal is unusually hydrophobic, and we established that altering this characteristic by making three point mutations near the signal peptidase cleavage site in MVE prM dramatically increased the extent of cleavage without requiring removal of the C protein. In addition, we demonstrated that luminal sequences downstream from the signal peptidase cleavage site contributed to the inefficiency of cleavage.
Antisense Downregulation of N-
myc1
in Woodchuck Hepatoma Cells Reverses the Malignant Phenotype
Authors: Wang Hai-Ping , Zhang Lunli , Dandri Maura , Rogler Charles E. 
Published: December 2019

ABSTRACT

Cell line WH44KA is a highly malignant woodchuck hepatoma cell line. WH44KA cells contain a single woodchuck hepatitis virus (WHV) DNA integration in the 3′ untranslated region of exon 3 of the woodchuck N-
myc1
gene. The highly rearranged WHV DNA contains WHV enhancers which activate the N-
myc
promoter, and a hybrid N-
myc1
–WHV mRNA is produced, which leads to a high steady-state level of N-myc1 protein. To investigate whether continuous N-
myc1
expression is required to maintain the tumor phenotype, we knocked out N-
myc
expression using a WHV–N-
myc1
antisense vector. We identified two WH44KA antisense cell lines, designated 4-5 and 4-11, in which steady-state N-myc1 protein levels were reduced by 95 and 80%, respectively. The growth rates of both antisense cell lines were reduced in comparison to those of wild-type and vector controls. The phenotype of 4-5 and 4-11 cells changed to a flattened appearance, and the cells exhibited contact inhibition. Colony-forming ability in soft agar was reduced by 92% for 4-5 cells and by 88% for 4-11 cells. Cell line 4-11 formed only small, slow-growing tumors in nude mice, consistent with a low level of N-myc1 remaining in the cells. In contrast, 4-5 cells, in which N-myc protein was reduced by greater than 95%, failed to form tumors in nude mice. The integrated WHV DNA contained the complete WHV X gene (WHx) and its promoter; however, we did not detect any WHx protein in the cells by using a sensitive assay. These data demonstrate that N-
myc
overexpression is required to maintain the malignant phenotype of WH44KA woodchuck hepatoma cells and provide a direct function for integrated WHV DNA in hepatocarcinogenesis.
Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus
Authors: Xiao Xiao , Li Juan , Samulski Richard Jude 
Published: December 2019

ABSTRACT

Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236–5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 10
5
viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.
Sequence Flexibility in the Polytropic
env
gp70-Derived Region of the Membrane Glycoprotein (gp55) of Friend Spleen Focus-Forming Virus Affects Its Biological Activity
Authors: Yugawa Takashi , Amanuma Hiroshi 
Published: December 2019

ABSTRACT

We previously reported (N. Watanabe, M. Nishi, Y. Ikawa, and H. Amanuma, J. Virol. 65:132–137, 1991) that the mutant Friend spleen focus-forming virus (F-SFFV
MS
), which encodes a mutant gp55 membrane glycoprotein with an ecotropic
env
gp70 sequence, was nonpathogenic. Here we injected the F-SFFV
MS
–Friend murine leukemia virus (F-MuLV) clone 57 complex into newborn DBA/2 mice. We obtained four groups of pathogenic variant F-SFFV complexes, each showing a different degree of pathogenicity in adult mice and a different gp55 profile. Of these, group 1 variant F-SFFV was particularly interesting, because it was the most frequently obtained and because it produced doublet bands of gp55 (59 and 57 kDa), neither of which reacted with the nonecotropic gp70-specific monoclonal antibody, and because its DNA intermediate did not hybridize with the nonecotropic
env
-specific probe. Cloning and DNA sequence analysis of the
env
region of one isolate of the group 1 variant F-SFFV revealed that this virus consisted of two distinct F-SFFV genomes; one (clone 117) differed from the other (clone 118) due to the presence of a 39-bp in-frame deletion. Reconstitution to full-length F-SFFV genomes and a pathogenicity assay showed that each reconstituted F-SFFV was pathogenic, with clone 117 showing a higher degree of pathogenicity than clone 118. Both reconstituted F-SFFVs caused activation of the mouse erythropoietin receptor in the factor-independent cell proliferation assay, although much less efficiently than the wild-type polycythemia-inducing isolate F-SFFVp. Clone 118 produced a gp55 of 59 kDa, while clone 117 produced one of 57 kDa. Clone 118 had a substitution by the F-MuLV clone 57 gp70 sequence, indicating that it was derived from the F-SFFV
MS
env
gene by a homologous recombination with the F-MuLV clone 57
env
gene. The site of the 39-bp deletion in clone 117 corresponded to the portion of the clone 118 sequence which was unique to the ecotropic
env
genes. These results indicated the importance for the biological activity of gp55 of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic
env
genes.