bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation betweenMycobacterium farcinogenesandMycobacterium senegalenseStrains Based on 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences
Authors: Hamid Mohamed E. , Roth Andreas , Landt Olfert , Kroppenstedt Reiner M. , Goodfellow Michael , Mauch Harald
Published: July 2002

16S ribosomal DNA (rDNA) and 16S-23S internal transcribed spacer rDNA sequence analyses were performed onMycobacterium farcinogenesandM. senegalensestrains and 26 strains of other rapidly growing mycobacteria to investigate the phylogenetic structure of bovine farcy mycobacteria within theM. fortuitumcomplex.M. farcinogenesandM. senegalensewere indistinguishable in their 5"-end 16S rDNA but showed both considerable interspecies spacer sequence divergence and a high level of intraspecies sequence stability. A rapid detection assay using PCR and hybridization with species-specific probes was developed. The assay was specific among 46 species other thanM. farcinogenesandM. senegalenseand correctly identified allM. farcinogenesandM. senegalensestrains. PCR- and 16S-23S rDNA sequence-based detection will be a valuable approach for diagnosis of the causal agents of African bovine farcy in cattle.
Evidence for Nucleic Acid Binding Ability and Nucleosome Association of
Bombyx mori
Nucleopolyhedrovirus BRO Proteins
Authors: Zemskov Evgueni A. , Kang WonKyung , Maeda Susumu
Published: July 2002

The
Bombyx mori nucleopolyhedrovirus
(BmNPV) genome contains five related members of the
bro
gene family, all of which are actively expressed in infected BmN cells. Although their functions are unknown, their amino acid sequences contain a motif found in all known viral and prokaryotic single-stranded DNA binding proteins. To determine if they bind to nucleic acids, we fractionated the nuclei of BmNPV-infected BmN cells using a histone extraction protocol. We detected BRO-A, BRO-C, and BRO-D in the histone H1 fraction using anti-BRO antibodies. Micrococcal nuclease treatment released these BRO proteins from the chromatin fraction, suggesting their involvement in nucleosome structures. Chromatographic fractionation showed that BRO-A and/or BRO-C interacted with core histones. Expression of partial sequences of BRO-A proved that the N-terminal 80 amino acid residues were required for DNA binding activity. We also demonstrated that BmNPV BRO proteins underwent phosphorylation and ubiquitination followed by proteasome degradation, which may explain their distribution in the cytoplasm as well as the nucleus. We propose that BRO-A and BRO-C may function as DNA binding proteins that influence host DNA replication and/or transcription.
Identification of Immunodominant and Conformational Epitopes in the Capsid Protein of Hepatitis E Virus by Using Monoclonal Antibodies
Authors: Riddell Michaela A. , Li Fan , Anderson David A.
Published: July 2002

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289–300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in
Escherichia coli
and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2.1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.
Evolution of Swine H3N2 Influenza Viruses in the United States
Authors: Webby Richard J. , Swenson Sabrina L. , Krauss Scott L. , Gerrish Philip J. , Goyal Sagar M. , Webster Robert G.
Published: July 2002

During 1998, severe outbreaks of influenza were observed in four swine herds in the United States. This event was unique because the causative agents, H3N2 influenza viruses, are infrequently isolated from swine in North America. Two antigenically distinct reassortant viruses (H3N2) were isolated from infected animals: a double-reassortant virus containing genes similar to those of human and swine viruses, and a triple-reassortant virus containing genes similar to those of human, swine, and avian influenza viruses (N. N. Zhou, D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K.-J. Yoon, S. Krauss, and R. G. Webster, J. Virol. 73:8851–8856, 1999). Because the U.S. pig population was essentially naive in regard to H3N2 viruses, it was important to determine the extent of viral spread. Hemagglutination inhibition (HI) assays of 4,382 serum samples from swine in 23 states indicated that 28.3% of these animals had been exposed to classical swine-like H1N1 viruses and 20.5% had been exposed to the triple-reassortant-like H3N2 viruses. The HI data suggested that viruses antigenically related to the double-reassortant H3N2 virus have not become widespread in the U.S. swine population. The seroreactivity levels in swine serum samples and the nucleotide sequences of six additional 1999 isolates, all of which were of the triple-reassortant genotype, suggested that H3N2 viruses containing avian PA and PB2 genes had spread throughout much of the country. These avian-like genes cluster with genes from North American avian viruses. The worldwide predominance of swine viruses containing an avian-like internal gene component suggests that these genes may confer a selective advantage in pigs. Analysis of the 1999 swine H3N2 isolates showed that the internal gene complex of the triple-reassortant viruses was associated with three recent phylogenetically distinct human-like hemagglutinin (HA) molecules. Acquisition of HA genes from the human virus reservoir will significantly affect the efficacy of the current swine H3N2 vaccines. This finding supports continued surveillance of U.S. swine populations for influenza virus activity.
Substitutions at the Putative Receptor-Binding Site of an Encephalitic Flavivirus Alter Virulence and Host Cell Tropism and Reveal a Role for Glycosaminoglycans in Entry
Authors: Lee Eva , Lobigs Mario
Published: July 2002

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp
390
, which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp
390
on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly
390
, Ala
390
, and His
390
) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.
Reovirus-Induced G
2
/M Cell Cycle Arrest Requires ς1s and Occurs in the Absence of Apoptosis
Authors: Poggioli George J. , Keefer Christopher , Connolly Jodi L. , Dermody Terence S. , Tyler Kenneth L.
Published: July 2002

Serotype-specific differences in the capacity of reovirus strains to inhibit proliferation of murine L929 cells correlate with the capacity to induce apoptosis. The prototype serotype 3 reovirus strains Abney (T3A) and Dearing (T3D) inhibit cellular proliferation and induce apoptosis to a greater extent than the prototype serotype 1 reovirus strain Lang (T1L). We now show that reovirus-induced inhibition of cellular proliferation results from a G
2
/M cell cycle arrest. Using T1L × T3D reassortant viruses, we found that strain-specific differences in the capacity to induce G
2
/M arrest, like the differences in the capacity to induce apoptosis, are determined by the viral S1 gene. The S1 gene is bicistronic, encoding the viral attachment protein ς1 and the nonstructural protein ς1s. A ς1s-deficient reovirus strain, T3C84-MA, fails to induce G
2
/M arrest, yet retains the capacity to induce apoptosis, indicating that ς1s is required for reovirus-induced G
2
/M arrest. Expression of ς1s in C127 cells increases the percentage of cells in the G
2
/M phase of the cell cycle, supporting a role for this protein in reovirus-induced G
2
/M arrest. Inhibition of reovirus-induced apoptosis failed to prevent virus-induced G
2
/M arrest, indicating that G
2
/M arrest is not the result of apoptosis related DNA damage and suggests that these two processes occur through distinct pathways.
Concerted Action of Multiple
cis
-Acting Sequences Is Required for Rev Dependence of Late Human Immunodeficiency Virus Type 1 Gene Expression
Authors: Graf Marcus , Bojak Alexandra , Deml Ludwig , Bieler Kurt , Wolf Hans , Wagner Ralf
Published: July 2002

Based on the human immunodeficiency virus type 1 (HIV-1)
gag
gene, subgenomic reporter constructs have been established allowing the contributions of different
cis
-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimized
gag
gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.
Posttranslational Processing of Infected Cell Protein 22 Mediated by Viral Protein Kinases Is Sensitive to Amino Acid Substitutions at Distant Sites and Can Be Cell-Type Specific
Authors: Poon Alice P. W. , Ogle William O. , Roizman Bernard
Published: July 2002

Infected cell protein 22 (ICP22) is posttranslationally phosphorylated by the viral kinases encoded by U
S
3 and U
L
13 and nucleotidylylated by casein kinase II. In rabbit and rodent cells and in primary human fibroblasts infected with mutants from which the α22 gene encoding ICP22 had been deleted, a subset of late (γ
2
) gene products exemplified by U
L
38 and U
S
11 proteins are expressed at a reduced level, as measured by the accumulation of both mRNA and protein. The same phenotype was observed in cells infected with mutants lacking the U
L
13 gene. The focus of this report is on three serine- and threonine-rich domains of ICP22. Two of these domains are homologs located between residues 38 to 66 and 300 to 328. The third domain is near the carboxyl terminus and contains the sequence T374SS. The results were as follows. (i) Alanine substitutions in the amino-terminal homolog precluded the posttranslational processing of ICP22 in rabbit skin cells and in Vero cells but had no effect on the accumulation of either U
S
11 or U
L
38 protein. (ii) Alanine substitutions in the carboxyl-terminal homolog had no effect on posttranslational processing of ICP22 accumulating in Vero cells but precluded full processing of ICP22 accumulating in rabbit skin cells. The effect on accumulation of U
L
38 and U
S
11 proteins was insignificant in Vero cells and minimal in rabbit skin cells. (iii) Substitutions of alanine for the threonine and serines in the third domain precluded full processing of ICP22 and caused a reduction of accumulation of U
S
11 and U
L
38 proteins. These results indicate the following. (i) The posttranslational processing of ICP22 is sensitive to mutations within the domains of ICP22 tested and is cell-type dependent. (ii) Posttranslational processing of ICP22 is not required for accumulation of U
L
38 and U
S
11 proteins to the same level as that seen in cells infected with the wild-type virus. (iii) The T374SS sequence shared by ICP22 and the U
S
1.5 proteins is essential for the accumulation of a subset of γ
2
proteins exemplified by U
S
11 and U
L
38 and is the first step in mapping of the sequences necessary for optimal accumulation of U
S
11 and U
L
38 proteins.
Open Reading Frame S/L of Varicella-Zoster Virus Encodes a Cytoplasmic Protein Expressed in Infected Cells
Authors: Kemble George W. , Annunziato Paula , Lungu Octavian , Winter Ruth E. , Cha Tai-An , Silverstein Saul J. , Spaete Richard R.
Published: July 2002

We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3′ untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.
Enhancing B- and T-Cell Immune Response to a Hepatitis C Virus E2 DNA Vaccine by Intramuscular Electrical Gene Transfer
Authors: Zucchelli Silvia , Capone Stefania , Fattori Elena , Folgori Antonella , Di Marco Annalise , Casimiro Danilo , Simon Adam J. , Laufer Ralph , La Monica Nicola , Cortese Riccardo , Nicosia Alfredo
Published: July 2002

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8+T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.
Modulation of Major Histocompatibility Class II Protein Expression by Varicella-Zoster Virus
Authors: Abendroth Allison , Slobedman Barry , Lee Eunice , Mellins Elizabeth , Wallace Mark , Arvin Ann M.
Published: July 2002

We sought to investigate the effects of varicella-zoster virus (VZV) infection on gamma interferon (IFN-γ)-stimulated expression of cell surface major histocompatibility complex (MHC) class II molecules on human fibroblasts. IFN-γ treatment induced cell surface MHC class II expression on 60 to 86% of uninfected cells, compared to 20 to 30% of cells which had been infected with VZV prior to the addition of IFN-γ. In contrast, cells that were treated with IFN-γ before VZV infection had profiles of MHC class II expression similar to those of uninfected cell populations. Neither IFN-γ treatment nor VZV infection affected the expression of transferrin receptor (CD71). In situ and Northern blot hybridization of MHC II (MHC class II DR-α) RNA expression in response to IFN-γ stimulation revealed that MHC class II DR-α mRNA accumulated in uninfected cells but not in cells infected with VZV. When skin biopsies of varicella lesions were analyzed by in situ hybridization, MHC class II transcripts were detected in areas around lesions but not in cells that were infected with VZV. VZV infection inhibited the expression of Stat 1α and Jak2 proteins but had little effect on Jak1. Analysis of regulatory events in the IFN-γ signaling pathway showed that VZV infection inhibited transcription of interferon regulatory factor 1 and the MHC class II transactivator. This is the first report that VZV encodes an immunomodulatory function which directly interferes with the IFN-γ signal transduction via the Jak/Stat pathway and enables the virus to inhibit IFN-γ induction of cell surface MHC class II expression. This inhibition of MHC class II expression on VZV-infected cells in vivo may transiently protect cells from CD4+T-cell immune surveillance, facilitating local virus replication and transmission during the first few days of cutaneous lesion formation.
Identification and Characterization of the Helix-Destabilizing Activity of Rotavirus Nonstructural Protein NSP2
Authors: Taraporewala Zenobia F. , Patton John T.
Published: July 2002

The rotavirus nonstructural protein NSP2 self-assembles into homomultimers, binds single-stranded RNA nonspecifically, possesses a Mg
2+
-dependent nucleoside triphosphatase (NTPase) activity, and is a component of replication intermediates. Because these properties are characteristics of known viral helicases, we examined the possibility that this was also an activity of NSP2 by using a strand displacement assay and purified bacterially expressed protein. The results revealed that, under saturating concentrations, NSP2 disrupted both DNA-RNA and RNA-RNA duplexes; hence, the protein possesses helix-destabilizing activity. However, unlike typical helicases, NSP2 required neither a divalent cation nor a nucleotide energy source for helix destabilization. Further characterization showed that NSP2 displayed no polarity in destabilizing a partial duplex. In addition, helix destabilization by NSP2 was found to proceed cooperatively and rapidly. The presence of Mg
2+
and other divalent cations inhibited by approximately one-half the activity of NSP2, probably due to the increased stability of the duplex substrate brought on by the cations. In contrast, under conditions where NSP2 functions as an NTPase, its helix-destabilizing activity was less sensitive to the presence of Mg
2+
, suggesting that in the cellular environment the two activities associated with the protein, helix destabilization and NTPase, may function together. Although distinct from typical helicases, the helix-destabilizing activity of NSP2 is quite similar to that of the ςNS protein of reovirus and to the single-stranded DNA-binding proteins (SSBs) involved in double-stranded DNA replication. The presence of SSB-like nonstructural proteins in two members of the family
Reoviridae
suggests a common mechanism of unwinding viral mRNA prior to packaging and subsequent minus-strand RNA synthesis.
Human Cytomegalovirus US2 Endoplasmic Reticulum-Lumenal Domain Dictates Association with Major Histocompatibility Complex Class I in a Locus-Specific Manner
Authors: Gewurz Benjamin E. , Wang Evelyn W. , Tortorella Domenico , Schust Danny J. , Ploegh Hidde L.
Published: July 2002

The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.
Modulation of Transporter Associated with Antigen Processing (TAP)-Mediated Peptide Import into the Endoplasmic Reticulum by Flavivirus Infection
Authors: Momburg Frank , Müllbacher Arno , Lobigs Mario
Published: July 2002

In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules, flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (A. Müllbacher and M. Lobigs, Immunity 3:207–214, 1995). Here we show that during the early phase of infection with different flaviviruses, the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50%. TAP expression is unaltered during infection, and viral but not host macromolecular synthesis is required for enhanced peptide transport. This study is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus-infected cells accounts for the upregulation of MHC class I cell surface expression with the biological consequence of viral evasion of natural killer cell recognition.
Differential Incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and Major Histocompatibility Complex Class I and II Molecules into Human Immunodeficiency Virus Type 1 Virions and Microvesicles: Implications for Viral Pathogenesis and Immune Regulation
Authors: Esser Mark T. , Graham David R. , Coren Lori V. , Trubey Charles M. , Bess Julian W. , Arthur Larry O. , Ott David E. , Lifson Jeffrey D.
Published: July 2002

Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4
+
T cells long before a quantitative decline in circulating CD4
+
T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4
+
T cells remains unclear. Both direct effects of cytopathic infection of CD4
+
cells and indirect effects in which uninfected “bystander” cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.
An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety
Authors: Heil Marintha L. , Albee Alison , Strauss James H. , Kuhn Richard J.
Published: July 2002

Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.
Sindbis Virus Variant with a Deletion in the 6K Gene Shows Defects in Glycoprotein Processing and Trafficking: Lack of Complementation by a Wild-Type 6K Gene in
trans
Authors: Sanz Miguel Angel , Carrasco Luis
Published: July 2002

A Sindbis virus (SV) variant with a 6K gene partially deleted has been obtained. This SV Del6K virus is defective in the proteolytic processing of virus glycoprotein precursor, transport of glycoproteins to the plasma membrane, and plaque phenotype. A revertant virus (SV Del6K-revQ21L) containing a point mutation in the deleted 6K gene was isolated and characterized. SV Del6K-revQ21L has corrected the defects of proteolytic processing and transport of virus glycoproteins to the plasma membrane, but it still remains attenuated compared to wild-type (wt) SV, exhibiting defects in virus budding. Neither mutant nor revertant viruses are complemented by the coexpression in
trans
of a wt SV 6K gene.
Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors
Authors: Qari Shoukat H. , Magre Saema , Garcı́a-Lerma J. Gerardo , Hussain Althaf I. , Takeuchi Yasuhiro , Patience Clive , Weiss Robin A. , Heneine Walid
Published: July 2002

Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC
50
s and IC
90
s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC
50
s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culture-based, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC
50
s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine.
Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in
Drosophila
Cells
Authors: Miller David J. , Schwartz Michael D. , Ahlquist Paul
Published: July 2002

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of
Drosophila
DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.
Repair of a Rev-Minus Human Immunodeficiency Virus Type 1 Mutant by Activation of a Cryptic Splice Site
Authors: Verhoef Koen , Bilodeau Patricia S. , van Wamel Jeroen L. B. , Kjems Jørgen , Stoltzfus C. Martin , Berkhout Ben
Published: July 2002

We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the
tat
-
rev
region of this revertant virus identified a second-site mutation in
tat
that restored virus replication in the mutant background. This mutation activated a cryptic 5′ splice site (ss) that, when used in conjunction with the regular HIV 3′ ss #5, fuses the
tat
and
rev
reading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5′ ss by mutational inactivation of an adjacent exon splicing silencer element.
φX174 Genome-Capsid Interactions Influence the Biophysical Properties of the Virion: Evidence for a Scaffolding-Like Function for the Genome during the Final Stages of Morphogenesis
Authors: Hafenstein Susan , Fane Bentley A.
Published: July 2002

During the final stages of φX174 morphogenesis, there is an 8.5-Å radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, φX174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the φX174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to- virion transition.
Critical Role for Glial Cells in the Propagation and Spread of Lymphocytic Choriomeningitis Virus in the Developing Rat Brain
Authors: Bonthius Daniel J. , Mahoney Jolonda , Buchmeier Michael J. , Karacay Bahri , Taggard Derek
Published: July 2002

Inoculation of the neonatal rat with lymphocytic choriomeningitis virus (LCMV) results in the selective infection of several neuronal populations and in focal pathological changes. However, the pathway by which LCMV reaches the susceptible neurons has not been described, and the nature and time course of the pathological changes induced by the infection are largely unknown. This study examined the sequential migration of LCMV in the developing rat brain and compared the pathological changes among infected brain regions. The results demonstrate that astrocytes and Bergmann glia cells are the first cells of the brain parenchyma infected with LCMV and that the virus spreads across the brain principally via contiguous glial cells. The virus then spreads from glial cells into neurons. However, not all neurons are susceptible to infection. LCMV infects neurons in only four specific brain regions: the cerebellum, olfactory bulb, dentate gyrus, and periventricular region. The virus is then cleared from glial cells but persists in neurons. LCMV induces markedly different pathological changes in each of the four infected regions. The cerebellum undergoes an acute and permanent destruction, while the olfactory bulb is acutely hypoplastic but recovers fully with age. Neurons of the dentate gyrus are unaffected in the acute phase but undergo a delayed-onset mortality. In contrast, the periventricular region has neither acute nor late-onset cell loss. Thus, LCMV infects four specific brain regions in the developing brain by spreading from glial cells to neurons and then induces substantially different pathological changes with diverse time courses in each of the four infected regions.
Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3C
pro
Authors: Back Sung Hoon , Kim Yoon Ki , Kim Woo Jae , Cho Sungchan , Oh Hoe Rang , Kim Jung-Eun , Jang Sung Key
Published: July 2002

The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3C
pro
(and/or 3CD
pro
), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3C
pro
target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA.
Distribution of Spontaneous Mutants and Inferences about the Replication Mode of the RNA Bacteriophage φ6
Authors: Chao Lin , Rang Camilla U. , Wong Linda E.
Published: July 2002

When a parent virus replicates inside its host, it must first use its own genome as the template for replication. However, once progeny genomes are produced, the progeny can in turn act as templates. Depending on whether the progeny genomes become templates, the distribution of mutants produced by an infection varies greatly. While information on the distribution is important for many population genetic models, it is also useful for inferring the replication mode of a virus. We have analyzed the distribution of mutants emerging from single bursts in the RNA bacteriophage φ6 and find that the distribution closely matches a Poisson distribution. The match suggests that replication in this bacteriophage is effectively by a stamping machine model in which the parental genome is the main template used for replication. However, because the distribution deviates slightly from a Poisson distribution, the stamping machine is not perfect and some progeny genomes must replicate. By fitting our data to a replication model in which the progeny genomes become replicative at a given rate or probability per round of replication, we estimated the rate to be very low and on the on the order of 10
−4
. We discuss whether different replication modes may confer an adaptive advantage to viruses.
Activation of Nuclear Factor of Activated T Cells by Human T-Lymphotropic Virus Type 1 Accessory Protein p12
I
Authors: Albrecht Björn , D'Souza Celine D. , Ding Wei , Tridandapani Susheela , Coggeshall K. Mark , Lairmore Michael D.
Published: July 2002

Human T-lymphotropic virus type 1 (HTLV-1) is the agent of an aggressive malignancy of CD4
+
T lymphocytes, called adult T-cell lymphoma/leukemia, and is associated with numerous immune-mediated diseases. To establish infection, HTLV-1 must activate targeted T cells during early stages of infection. We recently demonstrated that the HTLV-1 accessory protein p12
I
is critical for persistent infection in vivo and for viral infectivity in quiescent primary lymphocytes, suggesting a role for p12
I
in lymphocyte activation. To test whether p12
I
modulates signaling pathways required for T-lymphocyte activation, we examined AP-1-, NF-κB-, and nuclear factor of activated T cells (NFAT)-driven reporter gene activity in p12
I
-expressing Jurkat T cells compared to vector-transfected control cells. HTLV-1 p12
I
specifically induced NFAT-mediated transcription approximately 20-fold in synergy with the Ras/mitogen-activated protein kinase pathway, but did not influence AP-1- or NF-κB-dependent gene expression. Inhibition of calcium-dependent signals by cyclosporin A, BAPTA-AM [glycine,
N
,
N
′-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-
N
-2-(acetyloxy)methoxy-2-oxoethyl]-[bis(acetyloxy)methyl ester], and a dominant negative mutant of NFAT2 abolished the p12
I
-mediated activation of NFAT-dependent transcription. In contrast, inhibition of phospholipase C-γ and LAT (linker for activation of T cells) did not affect p12
I
-induced NFAT activity. Importantly, p12
I
functionally substituted for thapsigargin, which selectively depletes intracellular calcium stores. Our data are the first to demonstrate a role for HTLV-1 p12
I
in calcium-dependent activation of NFAT-mediated transcription in lymphoid cells. We propose a novel mechanism by which HTLV-1, a virus associated with lymphoproliferative disease, dysregulates common T-cell activation pathways critical for the virus to establish persistent infection.
Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture
Authors: Pietschmann Thomas , Lohmann Volker , Kaul Artur , Krieger Nicole , Rinck Gabriele , Rutter Gabriel , Strand Dennis , Bartenschlager Ralf
Published: July 2002

The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.
The Influence of Some Reaction Conditions on the Course of the Electrolytic Preparation of Ammonium Peroxodisulfate
Authors: Balej Jan , Thumová Milada , Špalková Hana
Published: July 2002

As a continuation of the previous paper, the results of the examination on the influence of further operating parameters (anodic current density, anodic current concentration, molar ratio of the starting solutions, P0 = m0[H2SO4] / m0[(NH4)2SO4], conversion degree, a = m[(NH4)2S2O8] / m0[(NH4)2SO4], composition and form of the anodic material, nature and concentration of anodic additives) on the anodic current efficiency χ and cell voltage of the laboratory flow-through integral electrolyzer model of the monopolar type were presented and discussed. The obtained results may be used for the optimization of the operating conditions of the industrial persulfate electrolyzer of the monopolar as well as bipolar type, too.
"Zero" and "Off-Zero" Critical Concentrations in Solutions of Polydisperse Polymers with Very High Molar Masses
Authors: Šolc Karel , Dušek Karel , Koningsveld Ronald , Berghmans Hugo
Published: July 2002

Polymer solutions with a concentration-dependent interaction parameter g(ϕ) are known to have sometimes critical polymer concentrations ϕc converging to a non-zero value (a so-called "off-zero" limiting critical point (CP)), as the chain length, m, grows to infinity, rather than to zero as usual (a "zero" limiting CP). In this report the criteria for the existence of both types, known for binary solutions with a linear g = g0 + g1ϕ, are extended to cover polydisperse polymers with a quadratic interaction function g(ϕ). Its coefficients g2 and ∆g2 = g2 - g1 determine the number and type of limiting CPs. Accordingly, the plane g2, ∆g2 is divided into the regions I (a zero CP), II (an off-zero CP), and III (a zero + two off-zero CPs). The region II is restricted to the half-plane with ∆g2 < -1/6, whereas the other half-plane with ∆g2 > -1/6 is shared by I and III. By varying the interactions, two limiting CPs may be brought together and merged in a heterogeneous double limiting CP. Such instances define the boundaries between the regions: at the I/III line, two off-zero CPs merge, whereas at the II/III line an off-zero CP coincides with a zero CP. A first-order perturbation theory of the latter double CPs, and a second-order perturbation theory of single "zero" CPs are developed, enabling meaningful extrapolations of data on polymers with high but finite molar masses. The latter theory yields extrapolation formulas for determination of Η-temperature, taking into account the polymer polydispersity and the concentration dependence of g. Solutions of polyisobutene in diphenyl ether and, possibly, in benzene appear to present experimental examples of off-zero limiting critical concentrations.
Photochemical Hydroxylation of Salicylic Acid with Hydrogen Peroxide; Mechanistic Study of Substrate Sensitized Reaction
Authors: Lang Kamil , Brodilová Jiřina , Luňák Stanislav
Published: July 2002

The photochemical hydroxylation of salicylic acid (SA) by hydrogen peroxide proceeds via the same intermediate independently of the excited reactant (i.e. SA or H2O2). This conclusion is supported by the similar effect of pH on the quantum yields and on the isomer ratio of formed products: 2,3-dihydroxybenzoic acid (2,3-DHB) and 2,5-dihydroxybenzoic acid (2,5-DHB). The product ratio ([2,3-DHB]/[2,5-DHB]) is not affected by H2O2 concentration.