bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase
Authors: Liu Huanting , Naismith James H. , Hay Ronald T. 
Published: July 2002

ABSTRACT
Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase α family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3′-5′-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087–1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.
Containment of Simian Immunodeficiency Virus Infection: Cellular Immune Responses and Protection from Rechallenge following Transient Postinoculation Antiretroviral Treatment
Authors: Lifson Jeffrey D. , Rossio Jeffrey L. , Arnaout Ramy , Li Li , Parks Thomas L. , Schneider Douglas K. , Kiser Rebecca F. , Coalter Vicky J. , Walsh Geneva , Imming Robert J. , Fisher Bradley , Flynn Bernard M. , Bischofberger Norbert , Piatak Michael , Hirsch Vanessa M. , Nowak Martin A. , Wodarz Dominik 
Published: July 2002

ABSTRACTTo better understand the viral and host factors involved in the establishment of persistent productive infection by primate lentiviruses, we varied the time of initiation and duration of postinoculation antiretroviral treatment with tenofovir {9-[2-(R)-(phosphonomethoxy)propyl]adenine}while performing intensive virologic and immunologic monitoring in rhesus macaques, inoculated intravenously with simian immunodeficiency virus SIVsmE660. Postinoculation treatment did not block the initial infection, but we identified treatment regimens that prevented the establishment of persistent productive infection, as judged by the absence of measurable plasma viremia following drug discontinuation. While immune responses were heterogeneous, animals in which treatment resulted in prevention of persistent productive infection showed a higher frequency and higher levels of SIV-specific lymphocyte proliferative responses during the treatment period compared to control animals, despite the absence of either detectable plasma viremia or seroconversion. Animals protected from the initial establishment of persistent productive infection were also relatively or completely protected from subsequent homologous rechallenge. Even postinoculation treatment regimens that did not prevent establishment of persistent infection resulted in downmodulation of the level of plasma viremia following treatment cessation, compared to the viremia seen in untreated control animals, animals treated with regimens known to be ineffective, or the cumulative experience with the natural history of plasma viremia following infection with SIVsmE660. The results suggest that the host may be able to effectively control SIV infection if the initial exposure occurs under favorable conditions of low viral burden and in the absence of ongoing high level cytopathic infection of responding cells. These findings may be particularly important in relation to prospects for control of primate lentiviruses in the settings of both prophylactic and therapeutic vaccination for prevention of AIDS.
Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities
Authors: Kirshner Jessica R. , Lukac David M. , Chang Jean , Ganem Don 
Published: July 2002

ABSTRACT
Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.
Subgenomic Negative-Strand RNA Function during Mouse Hepatitis Virus Infection
Authors: Baric Ralph S. , Yount Boyd 
Published: July 2002

ABSTRACT
Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenomic-length positive- and negative-strand RNAs. The origin and function of the subgenomic negative-strand RNAs is controversial. In this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these RNAs function as important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of positive-strand synthesis. Using cycloheximide, which preferentially inhibits negative-strand and to a lesser extent positive-strand synthesis, we demonstrate that cycloheximide treatment equally inhibits full-length and subgenomic-length negative-strand synthesis. Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection.
Mucosal Delivery of Inactivated Influenza Vaccine Induces B-Cell-Dependent Heterosubtypic Cross-Protection against Lethal Influenza A H5N1 Virus Infection
Authors: Tumpey Terrence M. , Renshaw Mary , Clements John D. , Katz Jacqueline M. 
Published: July 2002

ABSTRACTInfluenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modifiedEscherichia coliheat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6−/−)- or β2-microglobulin (β2m−/−)-deficient mice, respectively. β2m−/−but not IgH-6−/−vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.
Novel Baculovirus Expression Vectors That Provide Sialylation of Recombinant Glycoproteins in Lepidopteran Insect Cells
Authors: Jarvis Donald L. , Howe Dale , Aumiller Jared J. 
Published: July 2002

ABSTRACT
This report describes novel baculovirus vectors designed to express mammalian β1,4-galactosyltransferase and α2,6-sialyltransferase genes at early times after infection. Sf9 cells infected with these viral vectors, unlike cells infected with a wild-type baculovirus, produced a sialylated viral glycoprotein during the late phase of infection. Thus, the two mammalian glycosyltransferases encoded by these viral vectors are necessary and sufficient for sialylation of a foreign glycoprotein in insect cells under the conditions used in this study. While some of the new baculovirus vectors described in this study produced less, one produced wild-type levels of infectious budded virus progeny.
A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection
Authors: Arazi T. , Shiboleth Y. M. , Gal-On A. 
Published: July 2002

ABSTRACT
Systematic deletion and peptide tagging of the amino-terminal domain (NT, ∼43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5′ end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.
Insertion of Capsid Proteins from Nonenveloped Viruses into the Retroviral Budding Pathway
Authors: Krishna Neel K. , Wills John W. 
Published: July 2002

ABSTRACT
Retroviral Gag proteins direct the assembly and release of virus particles from the plasma membrane. The budding machinery consists of three small domains, the M (membrane-binding), I (interaction), and L (late or “pinching-off”) domains. In addition, Gag proteins contain sequences that control particle size. For Rous sarcoma virus (RSV), the size determinant maps to the capsid (CA)-spacer peptide (SP) sequence, but it functions only when I domains are present to enable particles of normal density to be produced. Small deletions throughout the CA-SP sequence result in the release of particles that are very large and heterogeneous, even when I domains are present. In this report, we show that particles of relatively uniform size and normal density are released by budding when the size determinant and I domains in RSV Gag are replaced with capsid proteins from two unrelated, nonenveloped viruses: simian virus 40 and satellite tobacco mosaic virus. These results indicate that capsid proteins of nonenveloped viruses can interact among themselves within the context of Gag and be inserted into the retroviral budding pathway merely by attaching the M and L domains to their amino termini. Thus, the differences in the assembly pathways of enveloped and nonenveloped viruses may be far simpler than previously thought.
Herpes Simplex Virus Type 1 Latency-Associated Transcript Gene Promotes Neuronal Survival
Authors: Thompson R. L. , Sawtell N. M. 
Published: July 2002

ABSTRACT
A complex interaction has evolved between the host's peripheral nervous system (PNS) and herpes simplex virus type 1 (HSV-1). Sensory neurons are permissive for viral replication, yet the virus can also enter a latent state in these cells. The interplay of viral and neuronal signals that regulate the switch between the viral lytic and latent states is not understood. The latency-associated transcript (LAT) regulates the establishment of the latent state and is required for >65% of the latent infections established by HSV-1 (R. L. Thompson and N. M. Sawtell, J. Virol. 71:5432–5440, 1997). To further investigate how LAT functions, a 1.9-kb deletion that includes the entire LAT promoter and 827 bp of the 5′ end of the primary LAT mRNA was introduced into strain 17syn+. The wild-type parent, three independently derived deletion mutants, and two independently derived genomically rescued variants of the mutants were analyzed in a mouse ocular model. The number of latent sites established in trigeminal ganglion (TG) neurons was determined using a single-cell quantitative PCR assay for the viral genome on purified TG neurons. It was found that the LAT null mutants established ∼75% fewer latent infections than the number established by the parental strain or rescued variant. The reduced establishment phenotype of LAT null mutants was due at least in part to a dramatic increase in the loss of TG neurons in animals infected with the LAT mutants. Over half of the neurons in the TG were destroyed following infection with the LAT mutants, and this was significantly more than were lost following infection with wild type. This is the first demonstration that the HSV LAT locus prevents the destruction of sensory neurons. The death of these neurons did not appear to be the result of increased apoptosis as measured by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay. Animals latently infected with the LAT null mutants reactivated less frequently in vivo and this was consistent with the reduction in the number of neurons in which latency was established. Thus, one function of the LAT gene is to protect sensory neurons and enhance the establishment of latency in the PNS.
Bovine Herpesvirus 1 Tegument Protein VP22 Interacts with Histones, and the Carboxyl Terminus of VP22 Is Required for Nuclear Localization
Authors: Ren Xiaodi , Harms Jerome S. , Splitter Gary A. 
Published: July 2002

ABSTRACT
The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the α-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection.
Role of Interleukin-4 (IL-4), IL-12, and Gamma Interferon in Primary and Vaccine-Primed Immune Responses to Friend Retrovirus Infection
Authors: Dittmer Ulf , Peterson Karin E. , Messer Ron , Stromnes Ingunn M. , Race Brent , Hasenkrug Kim J. 
Published: July 2002

ABSTRACTThe immunological resistance of a host to viral infections may be strongly influenced by cytokines such as interleukin-12 (IL-12) and gamma interferon (IFN-γ), which promote T helper type 1 responses, and IL-4, which promotes T helper type 2 responses. We studied the role of these cytokines during primary and secondary immune responses against Friend retrovirus infections in mice. IL-4- and IL-12-deficient mice were comparable to wild-type B6 mice in the ability to control acute and persistent Friend virus infections. In contrast, more than one-third of the IFN-γ-deficient mice were unable to maintain long-term control of Friend virus and developed gross splenomegaly with high virus loads. Immunization with a live attenuated vaccine virus prior to challenge protected all three types of cytokine-deficient mice from viremia and high levels of spleen virus despite the finding that the vaccinated IFN-γ-deficient mice were unable to class switch from immunoglobulin M (IgM) to IgG virus-neutralizing antibodies. The results indicate that IFN-γ plays an important role during primary immune responses against Friend virus but is dispensable during vaccine-primed secondary responses.
C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly
Authors: Castón José R. , Martı́nez-Torrecuadrada Jorge L. , Maraver Antonio , Lombardo Eleuterio , Rodrı́guez José F. , Casal J. Ignacio , Carrascosa José L. 
Published: July 2002

ABSTRACT

Infectious bursal disease virus
(IBDV), a member of the
Birnaviridae
family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.
Neuroadapted Yellow Fever Virus 17D: Genetic and Biological Characterization of a Highly Mouse-Neurovirulent Virus and Its Infectious Molecular Clone
Authors: Chambers Thomas J. , Nickells Michael 
Published: July 2002

ABSTRACTA neuroadapted strain of yellow fever virus (YFV) 17D derived from a multiply mouse brain-passaged virus (Porterfield YF17D) was additionally passaged in SCID and normal mice. The virulence properties of this virus (SPYF) could be distinguished from nonneuroadapted virus (YF5.2iv, 17D infectious clone) by decreased average survival time in SCID mice after peripheral inoculation, decreased average survival time in normal adult mice after intracerebral inoculation, and occurrence of neuroinvasiveness in normal mice. SPYF exhibited more efficient growth in peripheral tissues of SCID mice than YF5.2iv, resulting in a more rapid accumulation of virus burden, but with low-titer viremia, at the time of fatal encephalitis. In cell culture, SPYF was less efficient in replication than YF5.2iv in all cell lines tested. The complete nucleotide sequence of SPYF revealed 29 nucleotide substitutions relative to YF5.2iv, and these were distributed throughout the genome. There were a total of 13 predicted amino acid substitutions, some of which correspond to known differences among the Asibi, French viscerotropic virus, French neurotropic vaccine, and YF17D vaccine strains. The envelope (E) protein contained five substitutions, within all three functional domains. Substitutions were also present in regions encoding the NS1, NS2A, NS4A, and NS5 proteins and in the 3′ untranslated region (UTR). Construction of YFV harboring all of the identified coding nucleotide substitutions and those in the 3′ UTR yielded a virus whose cell culture and pathogenic properties, particularly neurovirulence and neuroinvasiveness for SCID mice, generally resembled those of the original SPYF isolate. These findings implicate the E protein and possibly other regions of the genome as virulence determinants during pathogenesis of neuroadapted YF17D virus in mice. The determinants affect replication efficiency in both neural and extraneural tissues of the mouse and confer some limited host-range differences in cultured cells of nonmurine origin.