bluuebunny/crossref_metadata_2025_split huggingface dataset:
Importance of the N Terminus of Rous Sarcoma Virus Protease for Structure and Enzymatic Function
Authors: Schatz Gisela W. , Reinking Jeffrey , Zippin Jonathan , Nicholson Linda K. , Vogt Volker M. 
Published: July 2002

ABSTRACT
All retrovirus proteases (PRs) are homodimers, and dimerization is essential for enzymatic function. The dimer is held together largely by a short four-stranded antiparallel beta sheet composed of the four or five N-terminal amino acid residues and a similar stretch of residues from the C terminus. We have found that the enzymatic and structural properties of Rous sarcoma virus (RSV) PR are exquisitely sensitive to mutations at the N terminus. Deletion of one or three residues, addition of one residue, or substitution of alanine for the N-terminal leucine reduced enzymatic activity on peptide and protein substrates 100- to 1,000-fold. The purified mutant proteins remained monomeric up to a concentration of about 2 mg/ml, as determined by dynamic light scattering. At higher concentrations, dimerization was observed, but the dimer lacked or was deficient in enzymatic activity and thus was inferred to be structurally distinct from a wild-type dimer. The mutant protein lacking three N-terminal residues (ΔLAM), a form of PR occurring naturally in virions, was examined by nuclear magnetic resonance spectroscopy and found to be folded at concentrations where it was monomeric. This result stands in contrast to the report that a similarly engineered monomeric PR of human immunodeficiency virus type 1 is unstructured. Heteronuclear single quantum coherence spectra of the mutant at concentrations where either monomers or dimers prevail were nearly identical. However, these spectra differed from that of the dimeric wild-type RSV PR. These results imply that the chemical environment of many of the amide protons differed and thus that the three-dimensional structure of the ΔLAM PR mutant is different from that of the wild-type PR. The structure of this mutant protein may serve as a model for the structure of the PR domain of the Gag polyprotein and may thus give clues to the initiation of proteolytic maturation in retroviruses.
Proper Processing of Avian Sarcoma/Leukosis Virus Capsid Proteins Is Required for Infectivity
Authors: Xiang Yan , Thorick Rebekah , Vana Marcy L. , Craven Rebecca , Leis Jonathan 
Published: July 2002

ABSTRACT
The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430–6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.
A Ty1 Reverse Transcriptase Active-Site Aspartate Mutation Blocks Transposition but Not Polymerization
Authors: Uzun Ozcan , Gabriel Abram 
Published: July 2002

ABSTRACT

Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D
211
N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme.
Origin Binding Protein-Containing Protein-DNA Complex Formation at Herpes Simplex Virus Type 1 oriS: Role in oriS-Dependent DNA Replication
Authors: Isler Jennifer A. , Schaffer Priscilla A. 
Published: July 2002

ABSTRACT
Initiation of herpes simplex virus type 1 (HSV-1) DNA replication during productive infection of fibroblasts and epithelial cells requires attachment of the origin binding protein (OBP), one of seven essential virus-encoded DNA replication proteins, to specific sequences within the two viral origins, oriL and oriS. Whether initiation of DNA replication during reactivation of HSV-1 from neuronal latency also requires OBP is not known. A truncated protein, consisting of the C-terminal 487 amino acids of OBP, termed OBPC, is the product of the HSV UL8.5 gene and binds to origin sequences, although OBPC's role in HSV DNA replication is not yet clear. To characterize protein-DNA complex formation at oriS in cells of neural and nonneural lineage, we used nuclear extracts of HSV-infected nerve growth factor-differentiated PC12 and Vero cells, respectively, as the source of protein in gel shift assays. In both cell types, three complexes (complexes A, B, and C) which contain either OBP or OBPC were shown to bind specifically to a probe which contains the highest-affinity OBP binding site in oriS, site 1. Complex A was shown to contain OBPC exclusively, whereas complexes B and C contained OBP and likely other cellular proteins. By fine-mapping the binding sites of these three complexes, we identified single nucleotides which, when mutated, eliminated formation of all three complexes, or complexes B and C, but not A. In transient DNA replication assays, both mutations significantly impaired oriS-dependent DNA replication, demonstrating that formation of OBP-containing complexes B and C is required for efficient initiation of oriS-dependent DNA replication, whereas formation of the OBPC-containing complex A is insufficient for efficient initiation.
Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells
Authors: Liu Yu-Tsueng , Brazas Rob , Ganem Don 
Published: July 2002

ABSTRACT

Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in
trans
. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses.
Simian Immunodeficiency Virus in Which
nef
and U3 Sequences Do Not Overlap Replicates Efficiently In Vitro and In Vivo in Rhesus Macaques
Authors: Münch Jan , Adam Nadia , Finze Nathaly , Stolte Nicole , Stahl-Hennig Christiane , Fuchs Dietmar , Ten Haaft Peter , Heeney Jonathan L. , Kirchhoff Frank 
Published: July 2002

ABSTRACT

The
nef
genes of human immunodeficiency virus and simian immunodeficiency virus (SIV) overlap about 80% of the U3 region of the 3′ long terminal repeat (LTR) and contain several essential
cis
-acting elements (here referred to as the TPI region): a T-rich region, the polypurine tract, and attachment (
att
) sequences required for integration. We inactivated the TPI region in the
nef
reading frame of the pathogenic SIVmac239 clone (239wt) by 13 silent point mutations. To restore viral infectivity, intact
cis
-regulatory elements were inserted just downstream of the mutated
nef
gene. The resulting SIV genome contains U3 regions that are 384 bp shorter than the 517-bp 239wt U3 region. Overall, elimination of the duplicated Nef coding sequences truncates the proviral genome by 350 bp. Nonetheless, it contains all known coding sequences and
cis
-acting elements. The TPI mutant virus expressed functional Nef and replicated like 239wt in all cell culture assays and in vivo in rhesus macaques. Notably, these SIVmac constructs allow us to study Nef function in the context of replication-competent viruses without the restrictions of overlapping LTR sequences and important
cis
-acting elements. The genomes of all known primate lentiviruses contain a large overlap between
nef
and the U3 region. We demonstrate that this conserved genomic organization is not obligatory for efficient viral replication and pathogenicity.
Role of Middle T-Small T in the Lytic Cycle of Polyomavirus: Control of the Early-to-Late Transcriptional Switch and Viral DNA Replication
Authors: Chen Li , Fluck Michele M. 
Published: July 2002

ABSTRACT
A comparative analysis of the lytic cycle of wild-type polyomavirus and middle T and small T defective mutants was carried out in the A2 genetic background. The results contrast with those obtained in comparisons between the hr-t type and their middle-T small-T-producing partners as previously described (20). The A2-derived mutants were found to share the maturation defect previously described for the hr-t mutants. However, their defect in DNA replication was more acute, resulting in a 5- to 100-fold decrease in the accumulation of viral genomes. Furthermore, their gene expression pattern was affected. A2-derived mutants displayed an early defect resulting in a 4- to 16-h delay in the expression of large T, and an alteration of the early-to-late transcriptional switch. In wild-type A2 infection, this switch is characterized by a large increase in the accumulation of early transcripts followed by late transcripts after the appearance of middle T and small T proteins and the onset of viral DNA replication (L. Chen and M. M. Fluck, J. Virol. 75: 8368–8379, 2001). In the mutant infection, increases in both classes of transcripts were delayed and reduced, but the effect on early transcripts was more pronounced. As has been described previously for the hr-t mutants (E. Goldman, J. Hattori, and T. Benjamin, Cell 13:505–513, 1979), the magnitude of these defects depended upon experimental conditions. Experiments using cytosine β-arabinofuranoside to reduce genome amplification suggest that the effect of middle T-small T on the transcriptional switch is not solely mediated by the effect of these protein(s) on increasing the number of templates. These data provide the first direct demonstration of an effect of middle T and/or small T in the viral transcription pattern during viral infection. The results agree with previous results obtained with plasmid reporters and with our understanding that the downstream targets of the middle T signaling pathway include three transcription factors that have binding sites in the enhancer domain that play a key regulatory role in the expression of the viral genes.
Phosphorylation of the Herpes Simplex Virus Type 1 Origin Binding Protein
Authors: Isler Jennifer A. , Schaffer Priscilla A. 
Published: July 2002

ABSTRACT

The herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), the product of the
UL9
gene, is one of seven HSV-encoded proteins required for viral DNA replication. OBP performs multiple functions characteristic of a DNA replication initiator protein, including origin-specific DNA binding and ATPase and helicase activities, as well as the ability to interact with viral and cellular proteins involved in DNA replication. Replication initiator proteins in other systems, including those of other DNA viruses, are known to be regulated by phosphorylation; however, the role of phosphorylation in OBP function has been difficult to assess due to the low level of OBP expression in HSV-infected cells. Using a metabolic labeling and immunoprecipitation approach, we obtained evidence that OBP is phosphorylated during HSV-1 infection. Kinetic analysis of metabolically labeled cells indicated that the levels of OBP expression and phosphorylation increased at approximately 4 h postinfection. Notably, when expressed from a transfected plasmid, a recombinant baculovirus, or a recombinant adenovirus (AdOBP), OBP was phosphorylated minimally, if at all. In contrast, superinfection of AdOBP-infected cells with an OBP-null mutant virus increased the level of OBP phosphorylation approximately threefold, suggesting that HSV-encoded viral or HSV-induced cellular factors enhance the level of OBP phosphorylation. Using HSV mutants inhibited at sequential stages of the viral life cycle, we demonstrated that this increase in OBP phosphorylation is dependent on early protein synthesis and is independent of viral DNA replication. Based on gel mobility shift assays, phosphorylation does not appear to affect the ability of OBP to bind to the HSV origins.
Replication-Competent or Attenuated, Nonpropagating Vesicular Stomatitis Viruses Expressing Respiratory Syncytial Virus (RSV) Antigens Protect Mice against RSV Challenge
Authors: Kahn Jeffrey S. , Roberts Anjeanette , Weibel Carla , Buonocore Linda , Rose John K. 
Published: July 2002

ABSTRACT
Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVΔG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVΔG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVΔG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVΔG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.
Unique Temperature-Sensitive Defect in Vaccinia Virus Morphogenesis Maps to a Single Nucleotide Substitution in the A30L Gene
Authors: Szajner Patricia , Weisberg Andrea S. , Moss Bernard 
Published: July 2002

ABSTRACT
Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.
Role of Cyclin D3 in the Biology of Herpes Simplex Virus 1 ICP0
Authors: Van Sant Charles , Lopez Pascal , Advani Sunil J. , Roizman Bernard 
Published: July 2002

ABSTRACT
Earlier reports from this laboratory have shown that the promiscuous transactivator infected-cell protein 0 (ICP0) binds and stabilizes cyclin D3, that the binding site maps to aspartic acid 199 (D199), and that replacement of D199 with alanine abolishes binding and reduces the capacity of the mutant virus to replicate in quiescent cells or to cause mortality in mice infected by a peripheral site. The objective of this report was to investigate the role of cyclin D3 in the biology of ICP0. We report the following results. (i) Wild-type ICP0 activates cyclin D-dependent kinase 4 (cdk4) and stabilizes cyclin D1 although ICP0 does not interact with this cyclin. (ii) The D199A mutant virus (R7914) does not activate cdk4 or stabilize cyclin D1, and neither the wild-type nor the mutant virus activates cdk2. (iii) Early in infection of human embryonic lung (HEL) fibroblasts both wild-type and D199A mutant ICP0s colocalize with PML, and in these cells the ND10 nuclear structures are dispersed. Whereas wild-type ICP0 is transported to the cytoplasm between 3 and 9 h. after infection, ICPO containing the D199A substitution remains quantitatively in the nucleus. (iv) To examine the interaction of ICP0 with cyclin D3, we used a previously described mutant carrying a wild-type ICP0 but expressing cyclin D3 (R7801) and in addition constructed a virus (R7916) that was identical except that it carried the D199A-substituted ICP0. Early in infection with R7801, ICP0 colocalized with cyclin D3 in structures similar to those containing PML. At 3 h after infection, ICP0 was translocated to the cytoplasm whereas cyclin D3 remained in the nucleus. The translocation of ICP0 to the cytoplasm was accelerated in cells expressing cyclin D3 compared with that of ICP0 expressed by wild-type virus. In contrast, ICP0 carrying the D199A substitution remained in the nucleus and did not colocalize with cyclin D3. These studies suggest the following conclusions. (i) ICP0 brings to the vicinity of ND10 cyclin D3 and, in consequence, an activated cdk4. The metabolic events occurring at or near that structure and involving cyclin D3 cause the translocation of ICP0 to the cytoplasm. (ii) In the absence of the cyclin D3 binding site in ICP0, cyclin D3 is not brought to ND10, cyclin D is not stabilized, and the function responsible for the translocation of ICP0 is not expressed, and in quiescent HEL fibroblasts the yields of virus are reduced.
Computational Analysis of Retrovirus-Induced
scid
Cell Death
Authors: Daniel René , Litwin Samuel , Katz Richard A. , Skalka Anna Marie 
Published: July 2002

ABSTRACT

It was shown recently that retroviral infection induces integrase-dependent apoptosis (programmed cell death) in DNA-dependent protein kinase (DNA-PK)-deficient
scid
pre-B cell lines, and it has been proposed that retroviral DNA integration is perceived as DNA damage that is repairable by the DNA-PK-dependent nonhomologous end-joining pathway (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644–647, 1999). Very few infectious virions seem to be necessary to induce
scid
cell death. In this study, we used a modeling approach to estimate the number of integration events necessary to induce cell death of DNA-PK-deficient
scid
cells. Several models for integration-mediated cell killing were considered. Our analyses indicate that a single hit (integration event) is sufficient to kill a
scid
cell. Moreover, the closest fit between the experimental data and our computational simulations was achieved with a model in which the infected
scid
cell must pass through S phase to trigger apoptosis. This model is consistent with the findings that a single double-strand DNA break is sufficient to kill a cell deficient in DNA repair and illustrates the potential of a modeling approach to address quantitative aspects of virus-cell interactions.
Adeno-Associated Virus Type 2-Mediated Gene Transfer: Altered Endocytic Processing Enhances Transduction Efficiency in Murine Fibroblasts
Authors: Hansen Jonathan , Qing Keyun , Srivastava Arun 
Published: July 2002

ABSTRACTAdeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, nonpathogenic human parvovirus that is currently in use as a vector for human gene therapy. However, the transduction efficiency of AAV vectors in different cell and tissue types varies widely. In addition to the lack of expression of the viral receptor and coreceptors and the rate-limiting viral second-strand DNA synthesis, which have been identified as obstacles to AAV-mediated transduction, we have recently demonstrated that impaired intracellular trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J. Hansen, K. Qing, H. J. Kwon, C. Mah, and A. Srivastava, J. Virol. 74:992–996, 2000). In this report, we document that escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293. While virions were found in both early and late endosomes or lysosomes of infected 293 cells, they were localized predominantly to the early endosomes in NIH 3T3 cells. Moreover, treatment of cells with bafilomycin A1 (Baf), an inhibitor of the vacuolar H+-ATPase and therefore of endosomal-lysosomal acidification, decreased the transduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated transduction in general, virions were detected in late endosomes and lysosomes, and these cells became sensitive to Baf-mediated inhibition of transduction. Thus, HU treatment overcomes defective endocytic processing of AAV in murine fibroblasts. These studies provide insights into the underlying mechanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy.
PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells
Authors: Johnson Marc C. , Scobie Heather M. , Vogt Volker M. 
Published: July 2002

ABSTRACT
While baculovirus expression of Gag proteins from numerous retroviruses has led reliably to production of virus-like particles (VLPs), we observed that expression of Rous sarcoma virus Gag failed to produce VLPs. Transmission and scanning electron microscopy analysis revealed that the Gag protein reached the plasma membrane but was unable to correctly form particles. Addition of a myristylation signal had no effect on the budding defect, but deletion of the PR domain of Gag restored normal budding. The resulting VLPs were morphologically distinct from human immunodeficiency virus type 1 VLPs expressed in parallel.