bluuebunny/crossref_metadata_2025 huggingface dataset:
A Glutaredoxin, Encoded by the G4L Gene of Vaccinia Virus, Is Essential for Virion Morphogenesis
Authors: White Christine L. , Weisberg Andrea S. , Moss Bernard
Published: July 2002

Vaccinia virus encodes two glutaredoxins, O2L and G4L, both of which exhibit thioltransferase and dehydroascorbate reductase activities in vitro. Although O2L was previously found to be dispensable for virus replication, we now show that G4L is necessary for virion morphogenesis. RNase protection and Western blotting assays indicated that G4L was expressed at late times after infection and was incorporated into mature virus particles. Attempts to isolate a mutant virus with a deleted G4L gene were unsuccessful, suggesting that the protein was required for virus replication. This interpretation was confirmed by the construction and characterization of a conditional lethal recombinant virus with an inducible copy of the G4L gene replacing the original one. Expression of G4L was proportional to the concentration of inducer, and the amount of glutaredoxin could be varied from barely detectable to greater than normal amounts of protein. Immunogold labeling revealed that the induced G4L protein was associated with immature and mature virions and adjacent cytoplasmic depots. In the absence of inducer, the production of infectious virus was severely inhibited, though viral late protein synthesis appeared unaffected except for decreased maturation-dependent proteolytic processing of certain core components. Electron microscopy of cells infected under nonpermissive conditions revealed an accumulation of crescent membranes on the periphery of electron-dense globular masses but few mature particles. We concluded that the two glutaredoxin homologs encoded by vaccinia virus have different functions and that G4L has a role in virion morphogenesis, perhaps by acting as a redox protein.
Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases
Authors: Seybert Anja , van Dinten Leonie C. , Snijder Eric J. , Ziebuhr John
Published: July 2002

The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in
Escherichia coli
as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5′ single-stranded regions on the partial-duplex substrates, indicating a 5′-to-3′ polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.
Intracellular Hepatitis B Virus Nucleocapsids Survive Cytotoxic T-Lymphocyte-Induced Apoptosis
Authors: Pasquetto Valerie , Wieland Stefan , Chisari Francis V.
Published: July 2002

Following antigen recognition, hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) induce a necroinflammatory liver disease in HBV-transgenic mice. An early event in this process is CTL-dependent activation of apoptosis in a small fraction of HBV-positive hepatocytes. Here we show that cytoplasmic HBV nucleocapsids and their cargo of replicative DNA intermediates survive CTL-induced apoptosis of hepatocytes in vitro. These results suggest that destruction of infected cells per se is not sufficient to destroy the replicating HBV genome in infected tissue and that other events in addition to this process are required for viral clearance to occur.
Primary Envelopment of Pseudorabies Virus at the Nuclear Membrane Requires the UL34 Gene Product
Authors: Klupp Barbara G. , Granzow Harald , Mettenleiter Thomas C.
Published: July 2002

Primary envelopment of several herpesviruses has been shown to occur by budding of intranuclear capsids through the inner nuclear membrane. By subsequent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding into vesicles in the
trans
-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also detected in the envelope of virions in the perinuclear space but is undetectable in intracytoplasmic and extracellular enveloped virus particles. Conversely, the tegument protein UL49 is present in mature virus particles and absent from perinuclear virions. In the absence of the UL34 protein, acquisition of the primary envelope is blocked and neither virus particles in the perinuclear space nor intracytoplasmic capsids or virions are observed. However, light particles which label with the anti-UL49 serum are formed in the cytoplasm. We conclude that the UL34 protein is required for primary envelopment, that the primary envelope is biochemically different from the final envelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, we provide additional evidence for a two-step envelopment process in herpesviruses.
Activation of Transcription of the Human Cytomegalovirus Early UL4 Promoter by the Ets Transcription Factor Binding Element
Authors: Chen Jiping , Stinski Mark F.
Published: July 2002

The human cytomegalovirus (HCMV) early UL4 promoter has served as a useful model for studying the activation of early viral gene expression. Previous transient-transfection experiments detected
cis
-acting elements (the NF-Y site and site 2) upstream of the transcriptional start site (L. Huang and M. F. Stinski, J. Virol. 69:7612–7621, 1995). The roles of two of these sites, the NF-Y site and site 2, in the context of the viral genome were investigated further by comparing mRNA levels from the early UL4 promoter in human foreskin fibroblasts infected by recombinant viruses with either wild-type or mutant
cis
-acting elements. Steady-state mRNA levels from the UL4 promoter with a mutation in the NF-Y site were comparable to that of wild type. A mutation in an Elk-1 site plus putative IE86 protein binding sites decreased the steady-state mRNA levels compared to the wild type at early times after infection. Electrophoretic mobility shift assays and antibody supershifts detected the binding of cellular transcription factor Elk-1 to site 2 DNA with infected nuclear extracts but not with mock-infected nuclear extracts. The role of cellular transcription factors activated by the mitogen activated protein kinase/extracellular signal-regulated kinase pathway in activating transcription from early viral promoters is discussed.
Methylation of Transcription Factor Binding Sites in the Epstein-Barr Virus Latent Cycle Promoter Wp Coincides with Promoter Down-Regulation during Virus-Induced B-Cell Transformation
Authors: Tierney R. J. , Kirby H. E. , Nagra J. K. , Desmond J. , Bell A. I. , Rickinson A. B.
Published: July 2002

Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.
Thogoto Virus Matrix Protein Is Encoded by a Spliced mRNA
Authors: Kochs Georg , Weber Friedemann , Gruber Simone , Delvendahl Alexander , Leitz Caroline , Haller Otto
Published: July 2002

Thogoto virus
(THOV) is a tick-transmitted orthomyxovirus with a segmented, negative-stranded RNA genome. In this study, we investigated the coding strategy of RNA segment 6 and found that it contains 956 nucleotides and codes for the matrix (M) protein. The full-length cDNA contains a single, long reading frame that lacks a stop codon but has coding capacity for a putative 35-kDa protein. In contrast, the M protein of THOV has an apparent molecular mass of 29 kDa as assessed by polyacrylamide gel electrophoresis. Therefore, we investigated the possibility of posttranscriptional processing of segment 6 transcripts by reverse transcription-PCR and identified a spliced mRNA that contains a stop codon and is translated into the 29-kDa M protein. Interestingly, the nontemplated UGA stop codon is generated by the splicing event itself. Thus, the unusual M coding strategy of THOV resembles that of
Influenza C virus
.
Comparison of Viral Genomic RNA Sorting Mechanisms in Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Moloney Murine Leukemia Virus
Authors: Dorman Nijsje , Lever Andrew
Published: July 2002

Genomic RNA sorting between translation and packaging was examined for human immunodeficiency virus type 1 (HIV-1) and HIV-2 using actinomycin D and leptomycin B treatment. Both viruses behaved differently from a simple retrovirus under actinomycin D treatment. With leptomycin B, the lack of apparent functional separation between translation and packaging functions in lentiviruses was confirmed. HIV-2 RNA levels were more stable, but reverse transcriptase production declined similarly to HIV-1.
Translation and Replication of Human Rhinovirus Type 14 and Mengovirus in
Xenopus
Oocytes
Authors: Gamarnik Andrea V. , Böddeker Nina , Andino Raul
Published: July 2002

We have previously shown that
Xenopus
oocytes require coinjection of both poliovirus RNA and HeLa cell extracts to support a complete cycle of viral replication yielding high levels of infectious viral particles. This novel system provides a tool for identifying host factors and for biochemically dissect individual steps that lead to virus production. Here we demonstrate that
Xenopus
oocytes are able to support replication of other picornaviruses such as human rhinovirus 14 and mengovirus. Unlike poliovirus, microinjection of mengovirus RNA yields high viral titers (about 10
7
PFU/oocyte) without the need for coinjection of additional cell extracts. In contrast, formation of infectious rhinovirus particles requires coinjection of human cell extracts. We found that one of these human factors is required for efficient rhinovirus translation. Our findings uncover differences in the host factor requirements among members of the picornavirus family and provide the means to identify the human protein(s) involved in rhinovirus production.
Isolation of Herpes Simplex Virus Procapsids from Cells Infected with a Protease-Deficient Mutant Virus
Authors: Newcomb William W. , Trus Benes L. , Cheng Naiqian , Steven Alasdair C. , Sheaffer Amy K. , Tenney Daniel J. , Weller Sandra K. , Brown Jay C.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (
m
100) deficient in the maturational protease for which it was expected that procapsids—normally, short-lived intermediates—would accumulate in infected cells. Particles isolated from
m
100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0°C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-Å resolution from cryoelectron micrographs showed
m
100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with
m
100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant
ts
Prot.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature,
ts
Prot.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.
The Latency-Associated Transcript Gene Enhances Establishment of Herpes Simplex Virus Type 1 Latency in Rabbits
Authors: Perng Guey-Chuen , Slanina Susan M. , Yukht Ada , Ghiasi Homayon , Nesburn Anthony B. , Wechsler Steven L.
Published: July 2002

The latency-associated transcript (LAT) gene the only herpes simplex virus type 1 (HSV-1) gene abundantly transcribed during neuronal latency, is essential for efficient in vivo reactivation. Whether LAT increases reactivation by a direct effect on the reactivation process or whether it does so by increasing the establishment of latency, thereby making more latently infected neurons available for reactivation, is unclear. In mice, LAT-negative mutants appear to establish latency in fewer neurons than does wild-type HSV-1. However, this has not been confirmed in the rabbit, and the role of LAT in the establishment of latency remains controversial. To pursue this question, we inserted the gene for the enhanced green fluorescent protein (EGFP) under control of the LAT promoter in a LAT-negative virus (ΔLAT-EGFP) and in a LAT-positive virus (LAT-EGFP). Sixty days after ocular infection, trigeminal ganglia (TG) were removed from the latently infected rabbits, sectioned, and examined by fluorescence microscopy. EGFP was detected in significantly more LAT-EGFP-infected neurons than ΔLAT-EGFP-infected neurons (4.9% versus 2%,
P
< 0.0001). The percentages of EGFP-positive neurons per TG ranged from 0 to 4.6 for ΔLAT-EGFP and from 2.5 to 11.1 for LAT-EGFP (
P
= 0.003). Thus, LAT appeared to increase neuronal latency in rabbit TG by an average of two- to threefold. These results suggest that LAT enhances the establishment of latency in rabbits and that this may be one of the mechanisms by which LAT enhances spontaneous reactivation. These results do not rule out additional LAT functions that may be involved in maintenance of latency and/or reactivation from latency.
Elucidating the Essential Role of the A14 Phosphoprotein in Vaccinia Virus Morphogenesis: Construction and Characterization of a Tetracycline-Inducible Recombinant
Authors: Traktman Paula , Liu Ke , DeMasi Joseph , Rollins Robert , Jesty Sophy , Unger Beth
Published: July 2002

We have previously reported the construction and characterization of v
ind
H1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-β-
d
-thiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1-deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (
tet
R), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, v
ind
A14. In the absence of TET, v
ind
A14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of “empty” crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG- and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants.
An Antiviral Compound That Blocks Structural Transitions of Poliovirus Prevents Receptor Binding at Low Temperatures
Authors: Dove Alan W. , Racaniello Vincent R.
Published: July 2002

Drugs such as WIN51711 that inhibit picornavirus replication are thought to block poliovirus infectivity by binding to the capsid and preventing structural transitions required for uncoating. We examined the activity of WIN51711 at temperatures where capsid flexibility is thought to be decreased. Below 37°C, WIN51711 inhibits the binding of wild-type poliovirus to cells but does not affect the binding of a poliovirus mutant which is believed to undergo structural transitions more readily. These results suggest that the poliovirus capsid must undergo structural changes to bind to its cellular receptor.
Structure of Recombinant Rabies Virus Nucleoprotein-RNA Complex and Identification of the Phosphoprotein Binding site
Authors: Schoehn Guy , Iseni Frédéric , Mavrakis Manos , Blondel Danielle , Ruigrok Rob W. H.
Published: July 2002

Rabies virus nucleoprotein (N) was produced in insect cells, in which it forms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies virus N-RNA and from nucleocapsid in virus, we propose a low-resolution model for rabies virus N-RNA in the virus.
Interleukin-12 (IL-12) and IL-18 Are Important in Innate Defense against Genital Herpes Simplex Virus Type 2 Infection in Mice but Are Not Required for the Development of Acquired Gamma Interferon-Mediated Protective Immunity
Authors: Harandi Ali M. , Svennerholm Bo , Holmgren Jan , Eriksson Kristina
Published: July 2002

Using a combination of gene-targeted mice and neutralizing antibodies, we showed that interleukin-12 (IL-12) and IL-18 are important in the innate control of genital herpes simplex virus type 2 infection but were not found to be critical, either singly or in combination, for the development of a protective gamma interferon-mediated immune response.
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

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

Neuroadapted 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

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

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

Among 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

In 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

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

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

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

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

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

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

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.
A Novel Silencer Element in the Bovine Papillomavirus Type 4 Promoter Represses the Transcriptional Response to Papillomavirus E2 Protein
Authors: Vance Keith W. , Campo M. Saveria , Morgan Iain M.
Published: July 2002

The long control regions (LCRs) of mucosal epitheliotropic papillomaviruses have similar organizations: a promoter region, an enhancer region, and a highly conserved distribution of E2 DNA binding sites (C. Desaintes and C. Demeret, Semin. Cancer Biol. 7:339–347, 1996). The enhancer of these viruses is epithelial cell specific, as it fails to activate transcription from heterologous promoters in nonepithelial cell types (B. Gloss, H. U. Bernard, K. Seedorf, and G. Klock, EMBO J. 6:3735–3743, 1987). Using the bovine papillomavirus type 4 (BPV-4) LCR and a bovine primary cell system, we have shown previously that a level of epithelial specificity resides in a papillomavirus promoter region. The BPV-4 promoter shows an enhanced response to transcriptional activators in epithelial cells compared with that of fibroblasts (K. W. Vance, M. S. Campo, and I. M. Morgan, J. Biol. Chem. 274:27839–27844, 1999). A chimeric lcr/tk promoter suggests that the upstream BPV-4 promoter region determines the cell-type-selective response of this promoter in fibroblasts and keratinocytes. Promoter deletion analysis identified two novel repressor elements that are, at least in part, responsible for mediating the differential response of this promoter to upstream activators in fibroblasts and keratinocytes. One of these elements, promoter repressor element 2 (PRE-2), is conserved in position and sequence in the related mucosal epitheliotropic papillomaviruses, BPV-3 and BPV-6. PRE-2 functions in
cis
to repress the basal activity of the simian virus 40 promoter and binds a specific protein complex. We identify the exact nucleotides necessary for binding and correlate loss of binding with loss of transcriptional repression. We also incorporate these mutations into the BPV-4 promoter and demonstrate an enhanced response of the mutated promoter to E2 in fibroblasts. The DNA binding protein in the detected complex is shown to have a molecular mass of approximately 50 kDa. The PRE-2 binding protein represents a novel transcriptional repressor and regulator of papillomavirus transcription.
Arbovirus of Marine Mammals: a New Alphavirus Isolated from the Elephant Seal Louse,
Lepidophthirus macrorhini
Authors: La Linn May , Gardner Joy , Warrilow David , Darnell Grant A. , McMahon Clive R. , Field Ian , Hyatt Alex D. , Slade Robert W. , Suhrbier Andreas
Published: July 2002

A novel alphavirus was isolated from the louse
Lepidophthirus macrorhini
, collected from southern elephant seals,
Mirounga leonina
, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates with the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class. The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.