bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome Variability and Capsid Structural Constraints of Hepatitis A Virus
Authors: Sánchez Glòria , Bosch Albert , Pintó Rosa M. 
Published: December 2002

ABSTRACT

The number of synonymous mutations per synonymous site (

K
s

), the number of nonsynonymous mutations per nonsynonymous site (

K
a

), and the codon usage statistic (

N
c

) were calculated for several hepatitis A virus (HAV) isolates. While

K
s

was similar to those of poliovirus (PV) and foot-and-mouth disease virus (FMDV),

K
a

was 1 order of magnitude lower. The

N
c

parameter provides information on codon usage bias and decreases when bias increases. The

N
c

value in HAV was about 38, while in PV and FMDV, it was about 53. The emergence of 22 rare codons in front of 8 in PV and 7 in FMDV was detected. Most of the conserved rare codons of the P1 region were strategically located at the carboxy borders of β barrels and α helices, their potential function being the assurance of proper folding of the capsid proteins through a decrease in the translation speed. This strategic location was not observed for amino acids encoded by the conserved rare codons of the 3D region. The percentage of bases with low pairing number values was higher in the latter region, suggesting a role of the conserved rare codons in the maintenance of RNA structure. Many of the rare codons in HAV are among the most frequent in humans, unlike in PV or in FMDV. This fact may be explained by the lack of cellular shutoff in HAV. One hypothesis is that HAV has evolved in order to avoid competition with its host for cellular tRNAs.
Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein
Authors: Scheifele Lisa Z. , Rhoads Jonathan D. , Parent Leslie J. 
Published: December 2002

ABSTRACT
Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.
Promoter Sequences of Varicella-Zoster Virus Glycoprotein I Targeted by Cellular Transactivating Factors Sp1 and USF Determine Virulence in Skin and T Cells in SCIDhu Mice In Vivo
Authors: Ito Hideki , Sommer Marvin H. , Zerboni Leigh , He Hongying , Boucaud Dwayne , Hay John , Ruyechan William , Arvin Ann M. 
Published: December 2002

ABSTRACTVaricella-zoster virus (VZV) glycoprotein I is dispensable in cell culture but necessary for infection of human skin and T cells in SCIDhu mice in vivo. The gI promoter contains an activating upstream sequence that binds the cellular transactivators specificity factor 1 (Sp1) and upstream stimulatory factor (USF) and an open reading frame 29 (ORF29)-responsive element (29RE), which mediates enhancement by ORF29 DNA binding protein of immediate-early 62 (IE62)-induced transcription. Recombinants, rOKAgI-Sp1 and rOKAgI-USF, with two base pair substitutions in Sp1 or USF sites, replicated like rOKA in vitro, but infectivity of rOKAgI-Sp1 was significantly impaired in skin and T cells in vivo. A double mutant, rOKAgI-Sp1/USF, did not replicate in skin but yielded low titers of infectious virus in T cells. The repaired protein, rOKAgI:rep-Sp1/USF, was as infectious as rOKA. Thus, disrupting gI promoter sites for cellular transactivators altered VZV virulence in vivo, with variable consequences related to the cellular factor and the host cell type. Mutations in the 29RE of the gI promoter were made by substituting each of four 10-bp blocks in this region with a 10-bp sequence, GATAACTACA, that was predicted to interfere with enhancer effects of the ORF29 protein. One of these mutants, which was designated rOKAgI-29RE-3, had diminished replication in skin and T cells, indicating that ORF29 protein-mediated enhancement of gI expression contributes to VZV virulence. Mutations within promoters of viral genes that are nonessential in vitro should allow construction of recombinant herpesviruses that have altered virulence in specific host cells in vivo and may be useful for designing herpesviral gene therapy vectors and attenuated viral vaccines.
Plasma Membrane Topology of Syncytial Domains of Herpes Simplex Virus Type 1 Glycoprotein K (gK): the UL20 Protein Enables Cell Surface Localization of gK but Not gK-Mediated Cell-to-Cell Fusion
Authors: Foster Timothy P. , Alvarez Xavier , Kousoulas Konstantin G. 
Published: December 2002

ABSTRACT
Most spontaneously occurring mutations that cause extensive herpes simplex virus type 1 (HSV-1)-induced cell fusion are single amino acid changes within glycoprotein K (gK). Despite the strong genetic association of gK with virus-induced cell fusion, its direct involvement in cellular membrane fusion has been controversial, largely due to previously unsuccessful efforts to detect gK expression on virion and cellular surfaces. Recently, we showed that gK is expressed on HSV-1 virions and functioned in virus entry (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). To determine whether gK is expressed on cellular surfaces, as well as its membrane topology, we generated the recombinant viruses gKV5DI, gKV5DII, gKV5DIII, and gKV5DIVcontaining insertions of the V5 antigenic epitope within each of four domains of gK predicted to localize either in the cytoplasmic side or in the extracytoplasmic side of cellular membranes. Immunohistochemical and confocal microscopy analyses of infected cells showed that both wild-type and syncytial forms of gK were expressed on cell surfaces. Analysis of the topology of the V5-tagged gK revealed that gK domains I and IV were located extracellularly, whereas domains II and III were localized intracellularly. Transiently expressed gK failed to localize in cellular plasma membranes. In contrast, infection of gK-transfected cells with the gK-null virus ΔgK enabled expression of gK on cell surfaces, as well as gK-mediated membrane fusion. Transient-coexpression experiments revealed that the UL20 protein enabled cell surface expression of gK, but not gK-mediated cell-to-cell fusion, indicating that additional viral proteins are required for expression of the gK syncytial phenotype.
The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step
Authors: Qu Feng , Ren Tao , Morris T. Jack 
Published: December 2002

ABSTRACT

Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of
Turnip crinkle virus
(TCV) strongly suppresses PTGS. The
Agrobacterium
infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and double-stranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants.
Tomato Ringspot Virus
Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes
Authors: Han Sumin , Sanfaçon Hélène 
Published: December 2002

ABSTRACT

Replication of all known positive-strand RNA viruses occurs in replication complexes associated with intracellular membranes. The putative nucleoside triphosphate binding (NTB) protein of
Tomato ringspot virus
(ToRSV) contains a stretch of hydrophobic residues at its C terminus, suggesting that it may act as a membrane anchor for the replication complex. Anti-NTB antibodies detected two predominant proteins in membrane-enriched fractions (the 66-kDa NTB and 69-kDa NTB-VPg proteins) along with other, larger proteins. The proteins containing the NTB domain cofractionated with markers of the endoplasmic reticulum (ER) and with ToRSV-specific RNA-dependent RNA polymerase activity in sucrose gradients. ToRSV infection induced severe changes in the morphology of the ER in plants expressing an ER-targeted green fluorescent protein (ER-GFP), and proteins containing the NTB domain colocalized with ER-GFP in indirect immunofluorescence assays. The proteins containing the NTB domain have properties of integral membrane proteins. Proteinase K protection assays using purified membranes from infected plants revealed that although the central portion of the NTB domain is exposed to the cytoplasmic face of the membranes, an 8-kDa fragment, recognized by anti-VPg antibodies, is protected by the membranes. This fragment probably consists of the 3-kDa VPg and the 5-kDa stretch of hydrophobic residues at the C terminus of the NTB protein, suggesting a luminal location for the VPg in at least a portion of the molecules. These results provide evidence that proteins containing the NTB domain are transmembrane proteins associated with ER-derived membranes and support the hypothesis that one or several of the proteins containing the NTB domain anchor the replication complex to the ER.
Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of
Autographa californica
Multiple Nucleopolyhedrovirus in Poorly Permissive
Spodoptera littoralis
Cells
Authors: Lu Liqun , Du Quansheng , Chejanovsky Nor 
Published: December 2002

ABSTRACT

Infection of
Spodoptera littoralis
SL2 cells with the baculovirus
Autographa californica
multiple nucleopolyhedrovirus (Ac
M
NPV) results in apoptosis and low yields of viral progeny, in contrast to infection with
S. littoralis
nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with Ac
M
NPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue Ac
M
NPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for
S. littoralis
larvae compared to Ac
M
NPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene
ie0
. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the
ie1
gene, a major Ac
M
NPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the
ie0
promoter locus by insertion of the chloramphenical acetyltransferase (
cat
) gene yielded a new recombinant Ac
M
NPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable Ac
M
NPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant Ac
M
NPV bearing an extra copy of the
ie1
gene under the control of the
Drosophila hsp70
promoter. These results suggest that IE0 plays a role in the regulation of Ac
M
NPV infection and show, for the first time, that significant improvement in the ability of Ac
M
NPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.
Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes
Authors: Saunier Bertrand , Triyatni Miriam , Ulianich Luca , Maruvada Padma , Yen Paul , Kohn Leonard D. 
Published: December 2002

ABSTRACTWe used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.
Concordant Modulation of Neutralization Resistance and High Infectivity of the Primary Human Immunodeficiency Virus Type 1 MN Strain and Definition of a Potential gp41 Binding Site in gp120
Authors: Leavitt Maria , Park Eun Ju , Sidorov Igor A. , Dimitrov Dimiter S. , Quinnan, Gerald V. 
Published: December 2002

ABSTRACT

Efforts to develop a vaccine against human immunodeficiency virus type 1 (HIV-1) are complicated by resistance of virus to neutralization. The neutralization resistance phenotype of HIV-1 has been linked to high infectivity. We studied the mechanisms determining this phenotype using clones of the T-cell-line-adapted (TCLA) MN strain (MN-TCLA) and the neutralization-resistant, primary MN strain (MN-P). Mutations in the amino- and carboxy-terminal halves of gp120 and the carboxy terminus of gp41 contributed to the neutralization resistance, high-infectivity phenotype but depended upon sequences in the leucine zipper (LZ) domain of gp41. Among 23 clones constructed to map the contributing mutations, there was a very strong correlation between infectivity and neutralization resistance (
R
2
= 0.81;
P
< 0.0001). Mutations that distinguished the gp120s of MN-P and MN-TCLA clones were clustered in or near the CD4 and coreceptor binding sites and in regions distant from those binding sites. To test the hypothesis that some of these distant mutations may interact with gp41, we determined which of them contributed to high infectivity and whether those mutations modulated gp120-gp41 association in the context of MN-P LZ sequences. In one clone, six mutations in the amino terminus of gp120, at least four of which clustered closely on the inner domain, modulated infectivity. This clone had a gp120-gp41 association phenotype like MN-P: in comparison to MN-TCLA, spontaneous dissociation was low, and dissociation induced by soluble CD4 binding was high. These results identify a region of the gp120 inner domain that may be a binding site for gp41. Our studies clarify mechanisms of primary virus neutralization resistance.
The Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Has Broad Signaling Effects in Primary Effusion Lymphoma Cells
Authors: Cannon Mark , Philpott Nicola J. , Cesarman Ethel 
Published: December 2002

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV-8]) is a gamma-2-herpesvirus responsible for Kaposi's sarcoma as well as primary effusion lymphoma (PEL). KSHV is a lymphotropic virus that has pirated many mammalian genes involved in inflammation, cell cycle control, and angiogenesis. Among these is the early lytic viral G protein-coupled receptor (vGPCR), a homologue of the human interleukin-8 (IL-8) receptor. When expressed, vGPCR is constitutively active and can signal via mitogen- and stress-activated kinases. In certain models it activates the transcriptional potential of NF-κB and activator protein 1 (AP-1) and induces vascular endothelial growth factor (VEGF) production. Despite its importance to the pathogenesis of all KSHV-mediated disease, little is known about vGPCR activity in hematopoietic cells. To study the signaling potential and downstream effects of vGPCR in such cells, we have developed PEL cell lines that express vGPCR under the control of an inducible promoter. The sequences required for tetracycline-mediated induction were cloned into a plasmid containing adeno-associated virus type 2 elements to enhance integration efficiency. This novel plasmid permitted studies of vGPCR activity in naturally infected KSHV-positive lymphocytes. We show that vGPCR activates ERK-2 and p38 in PEL cells. In addition, it increases the transcription of reporter genes under the control of AP-1, NF-κB, CREB, and NFAT, a Ca2+-dependent transcription factor important to KSHV lytic gene expression. vGPCR also increases the transcription of KSHV open reading frames 50 and 57, thereby displaying broad potential to affect viral transcription patterns. Finally, vGPCR signaling results in increased PEL cell elaboration of KSHV vIL-6 and VEGF, two growth factors involved in KSHV-mediated disease pathogenesis.
Structurally Conserved Amino Acid W501 Is Required for RNA Helicase Activity but Is Not Essential for DNA Helicase Activity of Hepatitis C Virus NS3 Protein
Authors: Kim Jong Won , Seo Mi Young , Shelat Anang , Kim Chon Saeng , Kwon Tae Woo , Lu Hui-hua , Moustakas Demetri Theodore , Sun Jeonghoon , Han Jang H. 
Published: December 2002

ABSTRACT

Hepatitis C virus (HCV) is a positive-strand RNA virus that encodes a helicase required for viral replication. Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU)
8
has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative π-facial hydrogen bond between the 2′-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the π-facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.
Rapid Prion Neuroinvasion following Tongue Infection
Authors: Bartz Jason C. , Kincaid Anthony E. , Bessen Richard A. 
Published: December 2002

ABSTRACT

Food-borne transmission of prions can lead to infection of the gastrointestinal tract and neuroinvasion via the splanchnic and vagus nerves. Here we report that the transmission of transmissible mink encephalopathy (TME) is 100,000-fold more efficient by inoculation of prions into the tongues of hamsters than by oral ingestion. The incubation period following TME agent (hereinafter referred to as TME) inoculation into the lingual muscles was the shortest among the five nonneuronal routes of inoculation, including another intramuscular route. Deposition of the abnormal isoform of the prion protein, PrP
Sc
, was first detected in the tongue and submandibular lymph node at 1 to 2 weeks following inoculation of the tongue with TME. PrP
Sc
deposits in the tongue were associated with individual axons, and the initial appearance of TME in the brain stem was found in the hypoglossal nucleus at 2 weeks postinfection. At later time points, PrP
Sc
was localized to brain cell groups that directly project to the hypoglossal nucleus, indicating the transneuronal spread of TME. TME PrP
Sc
entry into the brain stem preceded PrP
Sc
detection in the rostral cervical spinal cord. These results demonstrate that TME can replicate in both the tongue and regional lymph nodes but indicate that the faster route of brain invasion is via retrograde axonal transport within the hypoglossal nerve to the hypoglossal nucleus. Topical application of TME to a superficial wound on the surface of the tongue resulted in a higher incidence of disease and a shorter incubation period than with oral TME ingestion. Therefore, abrasions of the tongue in livestock and humans may predispose a host to oral prion infection of the tongue-associated cranial nerves. In a related study, PrP
Sc
was detected in tongues following the intracerebral inoculation of six hamster-adapted prion strains, which demonstrates that prions can also travel from the brain to the tongue in the anterograde direction along the tongue-associated cranial nerves. These findings suggest that food products containing ruminant or cervid tongue may be a potential source of prion infection for humans.
Complement Regulation by Kaposi's Sarcoma-Associated Herpesvirus ORF4 Protein
Authors: Spiller O. Brad , Robinson Mairi , O'Donnell Elizabeth , Milligan Steven , Morgan B. Paul , Davison Andrew J. , Blackbourn David J. 
Published: December 2002

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) is associated with three types of human tumor: Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. The virus encodes a number of proteins that participate in disrupting the immune response, one of which was predicted by sequence analysis to be encoded by open reading frame 4 (ORF4). The predicted ORF4 protein shares homology with cellular proteins referred to as regulators of complement activation. In the present study, the transcription profile of the ORF4 gene was characterized, revealing that it encodes at least three transcripts, by alternative splicing mechanisms, and three protein isoforms. Functional studies revealed that each ORF4 protein isoform inhibits complement and retains a C-terminal transmembrane domain. Consistent with the complement-regulating activity, we propose to name the proteins encoded by the ORF4 gene collectively as KSHV complement control protein (KCP). KSHV ORF4 is the most complex alternatively spliced gene encoding a viral complement regulator described to date. KCP inhibits the complement component of the innate immune response, thereby possibly contributing to the in vivo persistence and pathogenesis of this virus.