bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Generation and Characterization of a Soluble Form of the Semliki Forest Virus Fusion Protein
Authors: Lu Yanping E. , Eng Christina H. , Shome Swati Ghosh , Kielian Margaret 
Published: July 2002

ABSTRACT
During infection of host cells, a number of enveloped animal viruses are known to produce soluble forms of viral membrane glycoproteins lacking the transmembrane domain. The roles of such soluble glycoproteins in viral life cycles are incompletely understood, but in several cases they are believed to modulate host immune response and viral pathogenesis. Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells through low-pH-dependent fusion and buds from the plasma membrane. Fusion is mediated by the E1 subunit of the SFV spike protein. Previous studies described the in vivo generation of E1s, a truncated soluble form of E1, under conditions in which budding is inhibited in mammalian host cells. We have here examined the properties of E1s generation and the biological activity of E1s. E1s cleavage required spike protein transport out of the endoplasmic reticulum and was independent of virus infection. Cell surface E1 efficiently acted as a precursor for E1s. E1s generation was strongly pH dependent in BHK cells, with optimal cleavage at a pH of ≤7.0, conditions that inhibited the budding of SFV but not the budding of the rhabdovirus vesicular stomatitis virus. The pH dependence of E1s production and SFV budding was unaffected by the stability of the spike protein dimer but was a function of the host cell. Similar to the intact virus and in vitro-generated E1 ectodomain, treatment of E1s at low pH in the presence of target membranes triggered specific acid-dependent conformational changes. Thus, under a variety of conditions, SFV-infected cells can produce a soluble form of E1 that is biologically active.
Genetic Control of Neuroadapted Sindbis Virus Replication in Female Mice Maps to Chromosome 2 and Associates with Paralysis and Mortality
Authors: Thach Dzung C. , Kleeberger Steven R. , Tucker Pamela C. , Griffin Diane E. 
Published: July 2002

ABSTRACTNeuroadapted Sindbis virus (NSV) infection of mice causes hindlimb paralysis and 100% mortality in the C57BL/6 mouse strain, while adults of the BALB/cBy mouse strain are resistant to fatal encephalomyelitis. Levels of viral RNA are higher in the brains of infected C57BL/6 mice than in BALB/cBy mice (D. C. Thach et al., J. Virol. 74:6156–6161, 2000). These phenotypic differences between the two strains allowed us to map genetic loci involved in mouse susceptibility to NSV and to find relationships between mortality, paralysis, and viral RNA levels. Analysis of percent mortality inH2-congenic and F1mice suggested that theH2locus, sex linkage, and imprinting were not involved in determining susceptibility and that resistance was partially dominant over susceptibility. Segregation analysis using CXB recombinant inbred (RI) mice indicated that the percent mortality was multigenic. Interval mapping detected a suggestive quantitative trait locus (QTL) on chromosome 2 near marker D2Mit447. Analysis of paralysis in the RI mice detected the same suggestive QTL. Viral RNA level in F1mice was intermediate. Interval mapping using viral RNA levels in RI mice detected a significant QTL near marker D2Mit447 that explained 69% of the genetic variance. This QTL was confirmed in F2 mice and was designated asNsv1. Viral RNA level, percent paralyzed, and percent mortality were linearly correlated (r= 0.8 to 0.9). These results indicate that mortality, paralysis, and viral RNA levels are related complex traits and thatNsv1controls early viral load and determines the likelihood of paralysis and death.
Varicella-Zoster Virus ORF47 Protein Serine Kinase: Characterization of a Cloned, Biologically Active Phosphotransferase and Two Viral Substrates, ORF62 and ORF63
Authors: Kenyon T. K. , Lynch J. , Hay J. , Ruyechan W. , Grose C. 
Published: July 2002

ABSTRACT
Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.
Tomato Spotted Wilt Virus Glycoproteins Exhibit Trafficking and Localization Signals That Are Functional in Mammalian Cells
Authors: Kikkert Marjolein , Verschoor Ad , Kormelink Richard , Rottier Peter , Goldbach Rob 
Published: July 2002

ABSTRACT
The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.
Several E4 Region Functions Influence Mammary Tumorigenesis by Human Adenovirus Type 9
Authors: Thomas Darby L. , Schaack Jerome , Vogel Hannes , Javier Ronald 
Published: July 2002

ABSTRACTAmong oncogenic adenoviruses, human adenovirus type 9 (Ad9) is unique in eliciting exclusively estrogen-dependent mammary tumors in rats and in not requiring viral E1 region transforming genes for tumorigenicity. Instead, studies with hybrid viruses generated between Ad9 and the closely related nontumorigenic virus Ad26 have roughly localized an Ad9 oncogenic determinant(s) to a segment of the viral E4 region containing open reading frame 1 (E4-ORF1), E4-ORF2, and part of E4-ORF3. Although subsequent findings have shown that E4-ORF1 codes for an oncoprotein essential for tumorigenesis by Ad9, it is not known whether other E4 region functions may similarly play a role in this process. We report here that new results with Ad9/Ad26 hybrid viruses demonstrated that the minimal essential Ad9 E4-region DNA sequences include portions of both E4-ORF1 and E4-ORF2. Investigations with Ad9 mutant viruses additionally showed that the E4-ORF1 protein and certain E4-ORF2 DNA sequences are necessary for Ad9-induced tumorigenesis, whereas the E4-ORF2 and E4-ORF3 proteins are not. In fact, the E4-ORF3 protein was found to antagonize this process. Also pertinent was that certain crucial nucleotide differences between Ad9 and Ad26 within E4-ORF1 and E4-ORF2 were found to be silent with respect to the amino acid sequences of the corresponding proteins. Furthermore, supporting a prominent role for the E4-ORF1 oncoprotein in Ad9-induced tumorigenesis, an E1 region-deficient Ad5 vector that expresses the Ad9 but not the Ad26 E4-ORF1 protein was tumorigenic in rats and, like Ad9, promoted solely mammary tumors. These findings argue that the E4-ORF1 oncoprotein is the major oncogenic determinant of Ad9 and that an undefined regulatory element(s) within the E4 region represents a previously unidentified second function likewise necessary for tumorigenesis by this virus.
Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection
Authors: Pult Irmgard , Abbott Nathan , Zhang Yong-Yuan , Summers Jesse 
Published: July 2002

ABSTRACTIn this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 × 10−5to 6 × 10−5per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 × 10−5to 4.5 × 10−5per nucleotide per generation.
Mapping B-Cell Epitopes of Hepatitis C Virus E2 Glycoprotein Using Human Monoclonal Antibodies from Phage Display Libraries
Authors: Bugli Francesca , Mancini Nicasio , Kang Chang-Yuil , Di Campli Cristiana , Grieco Antonella , Manzin Aldo , Gabrielli Armando , Gasbarrini Antonio , Fadda Giovanni , Varaldo Pietro E. , Clementi Massimo , Burioni Roberto 
Published: July 2002

ABSTRACT
Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.
Hemagglutinin 1-Specific Immunoglobulin G and Fab Molecules Mediate Postattachment Neutralization of Influenza A Virus by Inhibition of an Early Fusion Event
Authors: Edwards M. J. , Dimmock N. J. 
Published: July 2002

ABSTRACT
In standard neutralization (STAN), virus and antibody are reacted together before inoculation of target cells, and inhibition of almost any of the processes concerned in the early interaction of virus and cell, including inhibition of virus attachment to cell receptors, can be the cause of neutralization by a particular monoclonal antibody (MAb). To simplify the interpretation of antibody action, we carried out a study of postattachment neutralization (PAN), where virus is allowed to attach to target cells before neutralizing antibody is introduced. We used influenza virus A/PR/8/34 (H1N1) and monoclonal immunoglobulin G (IgG) molecules and their Fabs specific to antigenic sites Sb (tip), Ca2 (loop), and Cb (hinge) of the hemagglutinin 1 (HA1) protein. All IgGs and Fabs gave PAN, although with reduced efficiency compared with STAN. Thus, bivalent binding of antibody was not essential for PAN. By definition, none of these MAbs gave PAN by inhibiting virus attachment, and they did not elute attached virus from the target cell or inhibit endocytosis of virus. However, virus-cell fusion, as demonstrated by R18 fluorescence dequenching or hemolysis of red blood cells, was inhibited in direct proportion to neutralization and in a dose-dependent manner and was thus likely to be responsible for the observed neutralization. However, to get PAN, it was necessary to inhibit the activation of the prefusion intermediate, the earliest known form on the fusion pathway that is created when virus is incubated at pH 5 and 4°C. PAN antibodies may act by binding HA trimers in contact with the cell and/or trimers in the immediate vicinity of the virus-cell contact point and so inhibit the recruitment of additional receptor-HA complexes.
A New RNA Element Located in the Coding Region of a Murine Endogenous Retrovirus Can Functionally Replace the Rev/Rev-Responsive Element System in Human Immunodeficiency Virus Type 1 Gag Expression
Authors: Wodrich Harald , Bohne Jens , Gumz Ellen , Welker Reinhold , Kräusslich Hans-Georg 
Published: July 2002

ABSTRACT

Nuclear export of incompletely spliced RNAs is a prerequisite for retroviral replication. Complex retroviruses like human immunodeficiency virus (HIV) encode a viral transport factor (Rev), which binds to its target sequence on the RNA genome and directs it into the Crm-1-mediated export pathway. Other retroviruses, like Mason-Pfizer monkey virus, contain
cis
-acting constitutive RNA transport elements (CTE) which achieve nuclear export of intron-containing RNA via cellular transport factors. Here, we describe the identification and characterization of a novel
cis
-acting orientation-dependent RNA expression element in the coding region of the murine intracisternal A-type particle (IAP) MIA14. This IAP expression element (IAPE) can functionally replace the Rev system in the expression of HIV-1 Gag proteins but functions independently of Crm-1. The presence of this element is needed for the expression of the IAP Gag proteins, indicating its biological significance. The IAPE can be functionally replaced by placing a CTE on the MIA14 RNA, further supporting its role in mRNA export. Northern blot analysis revealed that total RNA, as well as cytoplasmic RNA, was increased when the element was present. The element was mapped to a predicted stem-loop structure in the 3′ part of the
pol
open reading frame. There was no overall homology between the IAPE and the CTE, but there was complete sequence identity between short putative single-stranded loops. Deletion of these loops from the IAPE severely reduced Rev-independent Gag expression.
Proteasome-Independent Disruption of PML Oncogenic Domains (PODs), but Not Covalent Modification by SUMO-1, Is Required for Human Cytomegalovirus Immediate-Early Protein IE1 To Inhibit PML-Mediated Transcriptional Repression
Authors: Xu Yixun , Ahn Jin-Hyun , Cheng Mingfei , apRhys Colette M. , Chiou Chuang-Jiun , Zong Jianchao , Matunis Michael J. , Hayward Gary S. 
Published: July 2002

ABSTRACT
Human cytomegalovirus (HCMV) major immediate-early protein IE1 is an abundant 72-kDa nuclear phosphoprotein that is thought to play an important role in efficient triggering of the lytic cycle, especially at low multiplicity of infection. The best-known properties of IE1 at present are its transient targeting to punctate promyelocytic leukemia protein (PML)-associated nuclear bodies (PML oncogenic domains [PODs] or nuclear domain 10 [ND10]), with associated displacement of the cellular PML tumor suppressor protein into a diffuse nucleoplasmic form and its association with metaphase chromosomes. Recent studies have shown that the targeting of PML (and associated proteins such as hDaxx) to PODs is dependent on modification of PML by ubiquitin-like protein SUMO-1. In this study, we provide direct evidence that IE1 is also covalently modified by SUMO-1 in both infected and cotransfected cells, as well as in in vitro assays, with up to 30% of the protein representing the covalently conjugated 90-kDa form in stable U373/IE1 cell lines. Lysine 450 was mapped as the major SUMO-1 conjugation site, but a point mutation of this lysine residue in IE1 did not interfere with its targeting to and disruption of the PODs. Surprisingly, unlike PML or IE2, IE1 did not interact with either Ubc9 or SUMO-1 in yeast two-hybrid assays, suggesting that some additional unknown intranuclear cofactors must play a role in IE1 sumoylation. Interestingly, stable expression of either exogenous PML or exogenous Flag-SUMO-1 in U373 cell lines greatly enhanced both the levels and rate of in vivo IE1 sumoylation during HCMV infection. Unlike the disruption of PODs by the herpes simplex virus type 1 IE110(ICP0) protein, the disruption of PODs by HCMV IE1 proved not to involve proteasome-dependent degradation of PML. We also demonstrate here that the 560-amino-acid PML1 isoform functions as a transcriptional repressor when fused to the GAL4 DNA-binding domain and that wild-type IE1 inhibits the repressor function of PML1 in transient cotransfection assays. Furthermore, both IE1(1-346) and IE1(L174P) mutants, which are defective in displacing PML from PODs, failed to inhibit the repression activity of PML1, whereas the sumoylation-negative IE1(K450R) mutant derepressed as efficiently as wild-type IE1. Taken together, our results suggest that proteasome-independent disruption of PODs, but not IE1 sumoylation, is required for efficient IE1 inhibition of PML-mediated transcriptional repression.
Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication
Authors: Friebe Peter , Lohmann Volker , Krieger Nicole , Bartenschlager Ralf 
Published: July 2002

ABSTRACT

Sequences in the 5′ and 3′ termini of plus-strand RNA viruses harbor
cis
-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family
Flaviviridae
, a 341-nucleotide-long nontranslated region (NTR) is located at the 5′ end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5′ NTR required for RNA replication. We show that deletions introduced into the 5′ terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5′ NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5′ NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5′ NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus.
Characterization of Recombinant Hepatitis A Virus Genomes Containing Exogenous Sequences at the 2A/2B Junction
Authors: Beard Michael R. , Cohen Lisette , Lemon Stanley M. , Martin Annette 
Published: July 2002

ABSTRACT

Hepatitis A virus (HAV) differs from other members of the family
Picornaviridae
in that the cleavage of the polyprotein at the 2A/2B junction, commonly considered to be the primary polyprotein cleavage by analogy with other picornaviruses, is mediated by 3C
pro
, the only proteinase encoded by the virus. However, it has never been formally demonstrated that the 2A/2B junction is the site of primary cleavage, and the actual function of the 2A sequence, which lacks homology with sequence of other picornaviruses, remains unknown. To determine whether 2A functions in
cis
as a precursor with the nonstructural proteins, we constructed dicistronic HAV genomes in which a heterologous picornaviral internal ribosome entry site was inserted at the 2A/2B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possible
cis
replication function spanning the 2A/2B junction. However, infectious virus was recovered from recombinant HAV genomes containing exogenous protein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C
pro
cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not dependent upon the length of the inserted sequence. An HAV recombinant containing a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were deleted from replicating viruses, but this did not result from homologous recombination at the flanking 3C
pro
cleavage sites, since the 5′ and 3′ segments of the inserted sequence were retained in the deletion mutants. These results indicate that the HAV polyprotein can tolerate an insertion at the 2A/2B junction and that the 2A polypeptide does not function in
cis
as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding sequences inserted at the 2A/2B junction are novel and potentially useful protein expression vectors.
Endogenous Type D Retrovirus in a Marsupial, the Common Brushtail Possum (
Trichosurus vulpecula
)
Authors: Baillie Gregory J. , Wilkins Richard J. 
Published: July 2002

ABSTRACT

We have sequenced and characterized an endogenous type D retrovirus, which we have named TvERV(D), from the genome of an Australian marsupial, the common brushtail possum (
Trichosurus vulpecula
). Intact TvERV(D)
gag, pro, pol
, and
env
open reading frames were detected in the possum genome. TvERV(D) was classified as a type D retrovirus, most closely related to those of Old World monkeys, New World monkeys, and mice, based on phylogenetic analyses and genetic organization. Approximately 30 TvERV(D) proviruses are present in the genomes of possums, as detected by Southern hybridization. However, variability in fragment patterns between possums was observed and suggests recent (or ongoing) retrotranspositional activity.