bluuebunny/crossref_metadata_2025 huggingface dataset:
The PPPY Motif of Human T-Cell Leukemia Virus Type 1 Gag Protein Is Required Early in the Budding Process
Authors: Le Blanc Isabelle , Prévost Marie-Christine , Dokhélar Marie-Christine , Rosenberg Arielle R.
Published: September 2002

Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.
The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces both a G
2
and a Mitotic Block
Authors: Mauser Amy , Holley-Guthrie Elizabeth , Simpson Dennis , Kaufmann William , Kenney Shannon
Published: September 2002

The Epstein-Barr virus immediate-early protein BZLF1 is a transcriptional activator that mediates the switch from latent to lytic infection. Here we demonstrate that BZLF1 induces both a G
2
block and a mitotic block in HeLa cells and inhibits chromosome condensation. While the G
2
block is associated with decreased cyclin B1 in host cells and can be rescued by overexpression of cyclin B1, the mechanism for the mitotic defect is as yet undetermined.
Sustained Peptide-Specific Gamma Interferon T-Cell Response in Rhesus Macaques Immunized with Human Immunodeficiency Virus
gag
DNA Vaccines
Authors: Caulfield Michael J. , Wang Su , Smith Jeffrey G. , Tobery Timothy W. , Liu Xu , Davies Mary-Ellen , Casimiro Danilo R. , Fu Tong-Ming , Simon Adam , Evans Robert K. , Emini Emilio A. , Shiver John
Published: September 2002

We examined the influence of dose and method of antigen delivery on the dynamics and durability of T-cell responses to candidate human immunodeficiency virus (HIV) vaccines. Codon-optimized sequences from the HIV
gag
gene were inserted into alternative DNA vaccine vectors to express the coding sequence with or without the tissue plasminogen activator leader sequence. We delivered the vaccines by intramuscular injection as plasmid DNA without adjuvant or as plasmid DNA formulated with a novel block copolymer adjuvant (CRL8623) and then monitored the ensuing T-cell responses by using a gamma interferon enzyme-linked immunospot assay. We demonstrated persistence of the cell-mediated immune (CMI) response in rhesus macaques for at least 18 months following a four-dose vaccination regimen. The plasmid vaccine, with or without CRL8623, was immunogenic in macaques; however, the form coadministered with adjuvant exhibited improved T-cell responses, with a bias toward more antigen-specific CD8
+
T cells. Finally, we examined the fine specificity of the T-cell response to the
gag
vaccines by testing the response of 23 vaccinated macaques to individual Gag 20-mer peptides. Collectively, the monkeys responded to 25 epitopes, and, on average, each monkey recognized a minimum of 2.7 epitopes. The results indicate that a broad and durable CMI response to HIV DNA vaccines can be induced in a relevant nonhuman primate model.
Immune Responses to the Major Capsid Protein during Parvovirus Infection of Rats
Authors: Ball-Goodrich Lisa J. , Paturzo Frank X. , Johnson Elizabeth A. , Steger Krista , Jacoby Robert O.
Published: September 2002

Rat virus (RV) is a common parvovirus of laboratory rodents which can disrupt rat-based research. Prenatal or perinatal infection can be pathogenic or lead to persistent infection, whereas infection of adult rats is typically self-limiting. Effects on the host immune system have been documented during RV infection, but little is known about immune responses necessary for viral clearance. Our studies were conducted to identify humoral and cellular responses to the predominant capsid protein, VP2, during experimental infection of adult rats. We observed VP2-specific proliferation, gamma interferon production, and an immunoglobulin G2a humoral response that is maintained for at least 35 days following RV infection. These results strongly suggest the induction of virus-specific Th1-mediated immunity.
Reversal by Dithiothreitol Treatment of the Block in Murine Leukemia Virus Maturation Induced by Disulfide Cross-Linking
Authors: Campbell Stephen , Oshima Masamichi , Mirro Jane , Nagashima Kunio , Rein Alan
Published: September 2002

We previously reported that if murine leukemia virus particles are produced in the presence of the mild oxidizing agent disulfide-substituted benzamide-2, they fail to undergo the normal process of virus maturation. We now show that treatment of these immature particles with a reducing agent (dithiothreitol) induces their maturation in vitro, as evidenced by proteolytic cleavage of Gag, Gag-Pol, and Env proteins and by their morphology. The identification of partial cleavage products in these particles suggests the sequence with which the cleavages occur under these conditions. This may be a useful experimental system for further analysis of retroviral maturation under controlled conditions in vitro.
First Molecular Evidence for the Existence of Distinct Fish and Snake Adenoviruses
Authors: Benkő Mária , Élő Péter , Ursu Krisztina , Ahne Winfried , LaPatra Scott E. , Thomson Darelle , Harrach Balázs
Published: September 2002

From adenovirus-like viruses originating from a fish and a snake species, a conserved part of the adenoviral DNA polymerase gene was PCR amplified, cloned and sequenced. Phylogenetic analysis showed that the snake adenovirus is closely related to the members of the proposed genus
Atadenovirus
, whereas the fish isolate seems to represent a separate cluster, likely a new genus.
A Hepatitis B Surface Antigen Mutant That Lacks the Antigenic Loop Region Can Self-Assemble and Interact with the Large Hepatitis Delta Antigen
Authors: O'Malley Brendan , Lazinski David
Published: September 2002

A novel hepatitis B virus surface antigen mutant harboring a deletion of most of the major antigenic loop region was competent for self-assembly and secretion. Although the mutant protein was competent for interaction with and incorporation of free large hepatitis delta antigen, it was partially defective in hepatitis delta virus RNP incorporation.
Detection and Analysis of Hepatitis C Virus Sequences in Cerebrospinal Fluid
Authors: Laskus Tomasz , Radkowski Marek , Bednarska Agnieszka , Wilkinson Jeffrey , Adair Debra , Nowicki Marek , Nikolopoulou Georgia B. , Vargas Hugo , Rakela Jorge
Published: September 2002

Hepatitis C virus (HCV) sequences were detected in cerebrospinal fluid (CSF) in 8 of 13 HCV-positive patients. In four patients harboring different virus strains in serum and peripheral blood mononuclear cells (PBMC), CSF-derived virus was similar to that found in PBMC, which suggests that PBMC could carry HCV into the brain.
Pregenomic RNA Is Required for Efficient Incorporation of Pol Polyprotein into Foamy Virus Capsids
Authors: Heinkelein Martin , Leurs Cordula , Rammling Matthias , Peters Katrin , Hanenberg Helmut , Rethwilm Axel
Published: September 2002

The foamy virus (FV) Pol polyprotein is translated independently of Gag from a spliced mRNA. This method of expression raises the question of how Pol is associated with the viral particle. Using a transient FV vector transfection system, it is shown that pregenomic RNA is required for efficient virion incorporation of functionally active Pol and that protein-protein interactions of Pol with Gag are not sufficient to complete particle assembly.
Asian Genotypes of JC Virus in Japanese-Americans Suggest Familial Transmission
Authors: Suzuki Makoto , Zheng Huai-Ying , Takasaka Tomokazu , Sugimoto Chie , Kitamura Tadaichi , Beutler Ernest , Yogo Yoshiaki
Published: September 2002

To examine the mode of JC virus (JCV) transmission, we collected urine samples from second- and third-generation Japanese-Americans in Los Angeles, Calif., whose parents and grandparents were all Japanese. From the urine samples of these Japanese-Americans, we mainly detected two subtypes (CY and MY) of JCV that are predominantly found among native Japanese. This finding provides support for the hypothesis that JCV is transmitted mainly within the family through long-term cohabitation.
Inhibition of Herpes Simplex Virus Replication by WAY-150138: Assembly of Capsids Depleted of the Portal and Terminase Proteins Involved in DNA Encapsidation
Authors: Newcomb William W. , Brown Jay C.
Published: September 2002

Studies were carried out to examine the mechanism of action of WAY-150138, a member of a novel group of thiourea compounds recently shown to inhibit replication of herpes simplex virus type 1 (HSV-1). Previous studies have shown that the drug acts by preventing DNA encapsidation and that resistant mutants map to

U
L

6
, the gene encoding the protein subunit of the portal complex through which DNA enters the capsid. We tested the idea that WAY-150138 acts by preventing the incorporation of DNA-packaging proteins into capsids as they are assembled. Capsids were isolated from HSV-1-infected, drug-treated cells and examined by Western immunoblotting for the presence of two packaging proteins, the portal subunit (U
L
6) and a candidate terminase subunit (U
L
15). The results showed that both proteins were depleted in the capsids, suggesting that WAY-150138 antagonizes DNA encapsidation by depriving capsids of packaging proteins during the assembly process.
Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3′-Terminal Sequences
Authors: Smith Robert M. , Walton Cherie M. , Wu Catherine H. , Wu George Y.
Published: September 2002

The 3′-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute
cis
-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3′ untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3′ terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3′-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3′-X region RNA in vitro.
Vaccinia Virus J1R Protein: a Viral Membrane Protein That Is Essential for Virion Morphogenesis
Authors: Chiu Wen-Ling , Chang Wen
Published: September 2002

Vaccinia virus
, a member of the poxvirus family, contains a conserved J1R open reading frame that encodes a late protein of 17.8 kDa. The 18-kDa J1R protein is associated mainly with the membrane fraction of intracellular mature virus particles. This study examines the biological function of J1R protein in the vaccinia virus life cycle. A recombinant vaccinia virus was constructed to conditionally express J1R protein in an isopropyl-β-
d
-galactopyranoside (IPTG)-inducible manner. When J1R is not expressed during vaccinia virus infection, the virus titer is reduced approximately 100-fold. In contrast, J1R protein is not required for viral gene expression, as indicated by protein pulse-labeling. J1R protein is also not required for DNA processing, as the resolution of the concatemer junctions of replicated viral DNA was detected without IPTG. A deficiency of J1R protein caused a severe delay in the processing of p4a and p4b into mature core proteins 4a and 4b, indicating that J1R protein participates in virion morphogenesis. Infected cells grown in the absence of IPTG contained very few intracellular mature virions in the cytoplasm, and enlarged viroplasm structures accumulated with viral crescents attached at the periphery. Abundant intermediate membrane structures of abnormal shapes were observed, and many immature virions were either empty or partially filled, indicating that J1R protein is important for DNA packaging into immature virions. J1R protein also coimmunoprecipited with A45R protein in infected cells. In summary, these results indicate that vaccinia virus J1R is a membrane protein that is required for virus growth and plaque formation. J1R protein interacts with A45R protein and performs an important role during immature virion formation in cultured cells.
Replication of a Cytopathic Strain of Bovine Viral Diarrhea Virus Activates PERK and Induces Endoplasmic Reticulum Stress-Mediated Apoptosis of MDBK Cells
Authors: Jordan Robert , Wang Lijuan , Graczyk Thomas M. , Block Timothy M. , Romano Patrick R.
Published: September 2002

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca
2+
homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the
Flaviviridae
family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2α, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.
In Vitro Intersubtype Recombinants of Human Immunodeficiency Virus Type 1: Comparison to Recent and Circulating In Vivo Recombinant Forms
Authors: Quiñones-Mateu Miguel E. , Gao Yong , Ball Sarah C. , Marozsan Andre J. , Abraha Awet , Arts Eric J.
Published: September 2002

The increased prevalence of human immunodeficiency virus type 1 (HIV-1) intersubtype recombinants (ISRs) is shaping HIV-1 evolution throughout the world and will have an impact on both therapeutic and vaccine strategies. This study was designed to generate and compare in vitro ISRs to those isolated from HIV-infected individuals throughout the world. Human peripheral blood mononuclear cells were dually infected with seven pairs of HIV-1 isolates from different subtypes (i.e., A to F). Recombinant crossover sites were mapped to specific regions in the envelope (
env
) gene by using a cloning-hybridization technique and subtype-specific probes. In vitro intersubtype recombination was at least twofold more frequent in the V1-to-V3 region than in any other
env
fragment, i.e., C1 to V1, V3 to V5, or V5 to gp41. Sequence and recombination site analyses suggested the C2
env
domain as a “hot region” for recombination and selection of replication-competent ISRs during the 15-day incubation. In addition to these regional preferences for
env
recombination, homopolymeric nucleotide tracts, i.e., sequences known to pause reverse transcriptase and promote template switching, were found in most in vitro crossover sites. ISRs, originating from recent dual infections and limited transmission events, partly retained this in vitro regional or sequence preference for recombination sites. However, a shift to crossover sites flanking the gp120-coding sequence was evident in the stable circulating recombinant forms of HIV-1. Based on these findings, HIV-1 recombinants generated from these dual infections may be used as a model for in vivo intersubtype recombination and for the design of various diagnostic assays and vaccine constructs.
Intramolecular Recombinations of Moloney Murine Leukemia Virus Occur during Minus-Strand DNA Synthesis
Authors: Li Ting , Zhang Jiayou
Published: September 2002

Retroviral recombination can occur between two viral RNA molecules (intermolecular) or between two sequences within the same RNA molecule (intramolecular). The rate of retroviral intramolecular recombination is high. Previous studies showed that, after a single round of replication, 50 to 60% of retroviral recombinations occur between two identical sequences within a Moloney murine leukemia virus-based vector. Recombination can occur at any polymerization step within the retroviral replication cycle. Although reverse transcriptase is assumed to contribute to the template switches, previous studies could not distinguish between changes introduced by host RNA polymerase II (Pol II) or by reverse transcriptase. A cell culture system has been established to detect the individual contribution of host RNA Pol II, host DNA polymerase or viral reverse transcriptase, as well as the recombination events taking place during minus-strand DNA synthesis and plus-strand DNA synthesis in a single round of viral intramolecular replication. Studies in this report demonstrate that intramolecular recombination between two identical sequences during transcription by host RNA Pol II is minimal and that most recombinations occur during minus-strand DNA synthesis catalyzed by viral reverse transcriptase.
Feline Immunodeficiency Virus OrfA Is Distinct from Other Lentivirus Transactivators
Authors: Chatterji Udayan , de Parseval Aymeric , Elder John H.
Published: September 2002

The feline immunodeficiency virus (FIV) accessory factor, OrfA, facilitates transactivation of transcription directed by elements of the viral long terminal repeat (LTR). In order to map OrfA domains required for this transactivation, we used N- and C-terminal deletion constructs of the protein, expressed in a Gal4-based transactivation system. The results demonstrated that FIV OrfA, unlike other lentiviral transactivators such as visna virus Tat, is unable to transactivate from minimal promoter-based reporters and requires additional elements of the viral LTR. Stable CrFK-based cell lines were prepared that expressed OrfA to readily detectable levels and in which we were able to demonstrate 32-fold transactivation of an LTR-chloramphenicol acetyltransferase construct. Transactivation was heavily dependent on the presence of an ATF site within the viral LTR. Changing the translation initiation codon context substantially increased the level of production of OrfA from a bicistronic message that also encodes Rev. In the presence of a more favorable context sequence, the upstream expression of OrfA increased 21-fold, with only a 0.5-fold drop in downstream Rev expression. This suggests that Rev translation may occur via an internal ribosomal entry site rather than by leaky scanning.
Epstein-Barr Virus mRNA Export Factor EB2 Is Essential for Production of Infectious Virus
Authors: Gruffat Henri , Batisse Julien , Pich Dagmar , Neuhierl Bernhard , Manet Evelyne , Hammerschmidt Wolfgang , Sergeant Alain
Published: September 2002

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV
BMLF1-KO
). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV
BMLF1-KO
293 cells,
oriLyt
-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV
BMLF1-KO
mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.
Identification of Epstein-Barr Virus Strain Variants in Hairy Leukoplakia and Peripheral Blood by Use of a Heteroduplex Tracking Assay
Authors: Sitki-Green Diane , Edwards Rachel H. , Webster-Cyriaque Jennifer , Raab-Traub Nancy
Published: September 2002

Epstein-Barr virus (EBV) strains can be distinguished by specific sequence variations in the LMP1 gene. In this study, a heteroduplex tracking assay (HTA) specific for LMP1 was developed to precisely identify the prototypic undeleted strain B958, other undeleted strains (Ch2, AL, NC, and Med−), and strains with the 30-bp deletion (Med+ and Ch1). This technique also provides an estimate of the relative abundance of strains in patient samples. In this study, EBV strains were identified in 25 hairy leukoplakia (HLP) biopsies and six matched peripheral blood samples and throat washes with the LMP1-HTA. To investigate the relationship of the virus found in the peripheral blood to that in the HLP lesion, the strain variants in the peripheral blood B lymphocytes and those present within the epithelial cells in the HLP lesion and in throat washes were identified. In many of the subjects, compartmental differences in the EBV strain profiles in the oral cavity and peripheral blood were readily apparent. The throat wash specimens usually had a strain profile similar to that within the corresponding HLP sample, which was distinct from the strain profile detected in the peripheral blood. These analyses reveal that the nature of EBV infection can be very dynamic, with changes in relative strain abundance over time as well as the appearance of new strains. The patterns of abundance in the blood and oral cavity provide evidence for compartmentalization and for the transmission of strains between the blood and oropharynx.
Susceptibility to Polyomavirus-Induced Tumors in Inbred Mice: Role of Innate Immune Responses
Authors: Velupillai Palanivel , Carroll John P. , Benjamin Thomas L.
Published: September 2002

Mice of the PERA/Ei strain (PE mice) are highly susceptible to tumor induction by polyomavirus and transmit their susceptibility in a dominant manner in crosses with resistant C57BR/cdJ mice (BR mice). BR mice respond to polyomavirus infection with a type 1 cytokine response and develop effective cell-mediated immunity to the virus-induced tumors. By enumerating virus-specific CD8+T cells and measuring cytokine responses, we show that the susceptibility of PE mice is due to the absence of a type 1 cytokine response and a concomitant failure to sustain virus-specific cytotoxic T lymphocytes. (PE × BR)F1mice showed an initial type 1 response that became skewed toward type 2. Culture supernatants of splenocytes from infected PE mice stimulated in vitro contained high levels of interleukin-10 and no detectable gamma interferon, while those from BR mice showed the opposite pattern. Differences in the innate immune response to polyomavirus by antigen-presenting cells in PE mice and BR mice led to polarization of T-cell cytokine responses. Adherent cells from spleens of infected BR mice produced high levels of interleukin-12, while those from infected PE and F1mice produced predominantly interleukin-10. PE and F1mice infected by polyomavirus responded with increases in antigen-presenting cells expressing B7.2 costimulatory molecules, whereas BR mice responded with increased expression of B7.1. Administration of recombinant interleukin-12 along with virus resulted in partial protection of PE mice and provided complete protection against tumor development in F1animals.
The Mengovirus Leader Protein Suppresses Alpha/Beta Interferon Production by Inhibition of the Iron/Ferritin-Mediated Activation of NF-κB
Authors: Zoll Jan , Melchers Willem J. G. , Galama Jochem M. D. , van Kuppeveld Frank J. M.
Published: September 2002

In our studies on the biological function of the mengovirus leader protein, we identified a casein kinase II (CK-2) phosphorylation site in the protein. Here we report that the mengovirus leader protein can be phosphorylated by CK-2 in vitro. Expression of a recombinant leader protein in which the consensus CK-2 sequence around threonine 47 was disturbed resulted in a mutant protein that could no longer be phosphorylated. The CK-2 consensus sequence was modified by site-directed mutagenesis and subsequently introduced into a mengovirus cDNA clone to investigate the effect of the phosphorylation of the leader protein on virus replication and on the host cell response. Modifications by which the CK-2 consensus sequence was disturbed resulted in mutant viruses with reduced growth kinetics. We demonstrated that the integrity of the CK-2 phosphorylation site of the mengovirus leader protein was specifically related to the suppression of NF-κB activation and subsequent suppression of alpha/beta interferon production in infected cells. We also found that the integrity of the CK-2 phosphorylation site of the leader protein coincided with an increase of ferritin expression in the infected cell. These data indicate that the leader protein suppresses the iron-mediated activation of NF-κB and thereby inhibits alpha/beta interferon expression in the infected cell.
Relationship between SU Subdomains That Regulate the Receptor-Mediated Transition from the Native (Fusion-Inhibited) to the Fusion-Active Conformation of the Murine Leukemia Virus Glycoprotein
Authors: Lavillette Dimitri , Ruggieri Alessia , Boson Bertrand , Maurice Marielle , Cosset François-Loïc
Published: September 2002

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in
trans
during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the
trans
-acting RBD. Finally, we found mutations of the SU that bypass in
cis
the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in
cis
or in
trans
, between the receptor-activated RBD and a median segment of the freed C-terminal domain.
Identification and Characterization of a
cis
-Acting Replication Element (
cre
) Adjacent to the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus
Authors: Mason Peter W. , Bezborodova Svetlana V. , Henry Tina M.
Published: September 2002

Over the last few years, an essential RNA structure known as the
cis
-acting replicative element (
cre
) has been identified within the protein-coding region of several picornaviruses. The
cre
, a stem-loop structure containing a conserved AAACA motif, functions as a template for addition of U residues to the protein primer 3B. By surveying the genomes of representatives of several serotypes of foot-and-mouth disease virus (FMDV), we discovered a putative
cre
in the 5′ untranslated region of the genome (contiguous with the internal ribosome entry site [IRES]). To confirm the role of this putative
cre
in replication, we tested the importance of the AAACA motif and base pairing in the stem in FMDV genome replication. To this end,
cre
mutations were cloned into an FMDV replicon and into synthetic viral genomes. Analyses of the properties of these replicons and genomes revealed the following. (i) Mutations in the AAACA motif severely reduced replication, and all viruses recovered from genomes containing mutated AAACA sequences had reverted to the wild-type sequence. (ii) Mutations in the stem region showed that the ability to form this base-paired structure was important for replication. Although the
cre
was contiguous with the IRES, the mutations we created did not significantly reduce IRES-mediated translation in vivo. Finally, the position of the
cre
at the 5′ end of the genome was shown not to be critical for replication, since functional replicons and viruses lacking the 5′
cre
could be obtained if a wild-type
cre
was added to the genome following the 3D
pol
coding region. Taken together, these results support the importance of the
cre
in replication and demonstrate that the activity of this essential element does not require localization within the polyprotein-encoding region of the genome.
Phylogenetic and Functional Analysis of the Bacteriophage P1 Single-Stranded DNA-Binding Protein
Authors: Bendtsen Jannick Dyrløv , Nilsson Anders S. , Lehnherr Hansjörg
Published: September 2002

Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium
Escherichia coli
. A phylogenetic analysis indicated that the P1
ssb
gene coexists with its
E. coli
counterpart as an independent unit and does not represent a recent acquirement of the phage. The P1 and
E. coli
SSB proteins are fully functionally interchangeable. SSB-P1 is nonessential for phage growth in an exponentially growing
E. coli
host, and it is sufficient to promote bacterial growth in the absence of the
E. coli
SSB protein. Expression studies showed that the P1
ssb
gene is transcribed only, in an
rpoS
-independent fashion, during stationary-phase growth in
E. coli
. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an
ssb
-null mutant when exposed to a bacterial host in the stationary phase. These results reconciled the observed evolutionary conservation with the seemingly redundant presence of
ssb
genes in many bacteriophages and conjugative plasmids.
The Adenovirus E3 RID Complex Protects Some Cultured Human T and B Lymphocytes from Fas-Induced Apoptosis
Authors: McNees Adrienne L. , Garnett C. T. , Gooding Linda R.
Published: September 2002

Human group C adenoviruses cause an acute infection in respiratory epithelia and establish a long-term or persistent infection, possibly in lymphocytes. The mechanism by which this persistence is maintained is unknown; however, it would require that persistently infected lymphocytes not be deleted. The adenovirus genome encodes proteins that prevent the immune system from eliminating the virus-infected cell, including the E3 receptor internalization and degradation (RID) complex. The RID complex prevents death of infected cells by blocking apoptosis initiated through death domain-containing receptors of the tumor necrosis factor receptor (TNFR) superfamily, including TNFR1 (L. R. Gooding, T. S. Ranheim, A. E. Tollefson, L. Aquino, P. Duerksen-Hughes, T. M. Horton, and W. S. Wold, J. Virol. 65:4114-4123, 1991), TNF-related apoptosis-inducing ligand receptors (TRAIL-R1 and -R2) (C. A. Benedict, P. S. Norris, T. I. Prigozy, J. L. Bodmer, J. A. Mahr, C. T. Garnett, F. Martinon, J. Tschopp, L. R. Gooding, and C. F. Ware, J. Biol. Chem. 276:3270-3278, 2001; A. E. Tollefson, K. Toth, K. Doronin, M. Kuppuswamy, O. A. Doronina, D. L. Lichtenstein, T. W. Hermiston, C. A. Smith, and W. S. Wold, J. Virol. 75:8875-8887, 2001), and Fas (J. Shisler, C. Yang, B. Walter, C. F. Ware, and L. R. Gooding, J. Virol. 71:8299-8306, 1997). Here, we test the ability of RID to protect human lymphocytes from apoptosis induced by ligation of Fas, a mechanism important for regulating lymphocyte populations. Using a retrovirus expressing RID to infect six human lymphocyte cell lines, we found that RID functions in the absence of other viral proteins to downregulate surface Fas on some, but not all, cell lines. Total cellular levels of Fas decrease as measured by Western blotting, and this loss of Fas correlates with protection from apoptosis induced by ligation of Fas in every cell line tested. Although in some cases, RID causes loss of only a fraction of surface Fas, the presence of RID completely blocks the immediate events downstream of Fas ligation (i.e., Fas-FADD association and caspase-8 cleavage) in susceptible cell lines. Nonetheless, the ability of RID to block Fas signaling is independent of the Fas signaling pathway used (type I or type II). Interestingly, among the four T-cell lines tested, RID caused loss of Fas in the two T-cell lines bearing a relatively immature phenotype, while having no activity in T cells with mature phenotypes. Collectively, these data suggest that RID functions to prevent apoptosis of some human lymphocytes by internalizing surface Fas receptors. It is possible that the expression of RID facilitates long-term infection by preventing Fas-mediated deletion of persistently infected lymphocytes.
Human Immunodeficiency Virus Type 1 (HIV-1) Accessory Protein Vpr Induces Transcription of the HIV-1 and Glucocorticoid-Responsive Promoters by Binding Directly to p300/CBP Coactivators
Authors: Kino Tomoshige , Gragerov Alexander , Slobodskaya Olga , Tsopanomichalou Maria , Chrousos George P. , Pavlakis George N.
Published: September 2002

The accessory Vpr protein of human immunodeficiency virus type 1 (HIV-1) is a promiscuous activator of viral and cellular promoters. We report that Vpr enhances expression of the glucocorticoid receptor-induced mouse mammary tumor virus (MMTV) promoter and of the Tat-induced HIV-1 long terminal repeat promoter by directly binding to p300/CBP coactivators. In contrast, Vpr does not bind to p/CAF or to members of the p160 family of nuclear receptor coactivators, such as steroid receptor coactivator 1a and glucocorticoid receptor (GR)-interacting protein 1. Vpr forms a stable complex with p300 and also interacts with the ligand-bound glucocorticoid receptor in vivo. Mutation analysis showed that the C-terminal part of Vpr binds to the C-terminal portion of p300/CBP within amino acids 2045 to 2191. The same p300 region interacts with the p160 coactivators and with the adenovirus E1A protein. Accordingly, E1A competed for binding to p300 in vitro. Coexpression of E1A or of small fragments of p300 containing the Vpr binding site resulted in inhibition of Vpr's transcriptional effects. The C-terminal part of p300 containing the transactivating region is required for Vpr transactivation, whereas the histone acetyltransferase enzymatic region is dispensable. Vpr mutants that bind p300 but not the GR did not activate expression of the MMTV promoter and had dominant-negative effects. These results indicate that Vpr activates transcription by acting as an adapter linking transcription components and coactivators.
Persistence of Borna Disease Virus in Naturally Infected Sheep
Authors: Vahlenkamp Thomas W. , Konrath Andrea , Weber Matthias , Müller Hermann
Published: September 2002

To get an insight into Borna disease virus (BDV) epidemiology, an isolated flock of approximately 25 sheep within the region of Southeast Germany to which the disease is endemic was investigated over a 3-year observation period. BDV-specific antibodies and RNA in peripheral blood mononuclear cells were detected in 12.5 (year 1), 11.5 (year 2), and 19.4% (year 3) and 1.6 (year 1), 0 (year 2), and 14.9% (year 3) of the animals, respectively. BDV persisted in asymptomatic sheep for up to 2 years. Significantly higher numbers of antibody-positive animals were detected seasonally in spring and early summer, the times when usually most of the clinical cases of Borna disease occur. In spring of the third year, numbers of antibody-positive and viral-RNA-positive animals increased significantly despite their having no obvious clinical symptoms. The removal of all antibody- and RNA-positive animals from the flock did not reduce the prevalence of BDV infections in the following year. During a 3-month observation period of three antibody-positive animals, viral RNA was repeatedly detected by reverse transcription-PCR in nasal secretions, saliva, and conjunctival fluids. Sequence analysis revealed clustered nucleotide exchanges among sheep BDV p24 genomes, which differed at five positions from the clustered nucleotide exchanges seen in horse BDV p24 genomes.
An Early Regulatory Function Required in a Cell Type-Dependent Manner Is Expressed by the Genomic but Not the cDNA Copy of the Herpes Simplex Virus 1 Gene Encoding Infected Cell Protein 0
Authors: Poon Alice P. W. , Silverstein Saul J. , Roizman Bernard
Published: September 2002

The α0 genes of herpes simplex virus 1 (HSV-1) contain three exons. Earlier studies have shown that the substitution of genomic sequences with a cDNA copy does not alter the capacity of the virus to replicate or establish latent infection. Other studies have demonstrated that HSV-1 may express alternatively spliced forms of α0 transcripts. The studies reported here centered on a mutant HSV-1(vCPc0) strain in which the genomic copies of the α0 gene were replaced with cDNA copies. From our research, we report the following observations. (i) In contrast to events transpiring in cells infected with wild-type virus, the expression of HSV-1(vCPc0) genes was delayed or restricted to α genes for many hours in rabbit skin cells and to a lesser extent in HEp-2 cells but not in Vero cells. This delay in the expression of HSV-1(vCPc0) β or γ genes was also multiplicity of infection dependent. (ii) Exposure to MG132, a proteasomal inhibitor, before infection with wild-type virus had no significant effect on the accumulation of viral proteins in Vero cells and caused an only slight delay in viral gene expression in rabbit skin cells in a multiplicity of infection-dependent fashion. The drug had no effect when it was added to the medium 3 h after infection. (iii) Rabbit skin or HEp-2 cells exposed to MG132 3 h after infection with the HSV-1(vCPc0) mutant accumulated only α proteins. This restriction was cell type dependent but not multiplicity of infection dependent. (iv) Both the delay in the expression of β and γ genes and the effect of MG132 added to the medium 3 h after infection were rescued by restoration of the intron 1 sequences in the HSV-1(vCPc0) mutant. However, cells transduced by baculoviruses expressing intron 1 RNA did not complement the HSV-1(vCPc0) mutant, suggesting that the function of intron 1 is in
cis
rather than in
trans
. We came to the following conclusions as a result. (i) Post-α gene expression requires the involvement of the proteasomal pathway in a cell type-dependent manner. Consistent with this requirement, the proapoptotic functions of MG132 are blocked in cells infected before exposure to the drug but not after exposure. (ii) A function encoded by the α0 gene that is absent from the cDNA copy is required for viral gene expression in a cell type- and multiplicity of infection-dependent fashion. The absence of this master function delays but does not ultimately block viral gene expression in the cell lines tested here.
A Conserved Capsid Protein Surface Domain of
Cucumber Mosaic Virus
Is Essential for Efficient Aphid Vector Transmission
Authors: Liu Sijun , He Xiaohua , Park Gyungsoon , Josefsson Caroline , Perry Keith L.
Published: September 2002

A prominent feature on the surfaces of virions of
Cucumber mosaic virus
(CMV) is a negatively charged loop structure (the βH-βI loop). Six of 8 amino acids in this capsid protein loop are highly conserved among strains of CMV and other cucumoviruses. Five of these amino acids were individually changed to alanine or lysine (an amino acid of opposite charge) to create nine mutants (the D191A, D191K, D192A, D192K, L194A, E195A, E195K, D197A, and D197K mutants). Transcripts of cDNA clones were infectious when they were mechanically inoculated onto tobacco, giving rise to symptoms of a mottle-mosaic typical of the wild-type virus (the D191A, D191K, D192A, E195A, E195K, and D197A mutants), a systemic necrosis (the D192K mutant), or an atypical chlorosis with necrotic flecking (the L194A mutant). The mutants formed virions and accumulated to wild-type levels, but eight of the nine mutants were defective in aphid vector transmission. The aspartate-to-lysine mutation at position 197 interfered with infection; the only recovered progeny (the D197K∗ mutant) harbored a second-site mutation (denoted by the asterisk) of alanine to glutamate at position 193, a proximal site in the βH-βI loop. Since the disruption of charged amino acid residues in the βH-βI loop reduces or eliminates vector transmissibility without grossly affecting infectivity or virion formation, we hypothesize that this sequence or structure has been conserved to facilitate aphid vector transmission.
Hepatitis B Virus X Protein Activates the p38 Mitogen-Activated Protein Kinase Pathway in Dedifferentiated Hepatocytes
Authors: Tarn Chi , Zou Lin , Hullinger Ronald L. , Andrisani Ourania M.
Published: September 2002

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca
2+
and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G
2
/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.
The Vaccinia Virus E8R Gene Product: a Viral Membrane Protein That Is Made Early in Infection and Packaged into the Virions' Core
Authors: Doglio Laura , De Marco Ario , Schleich Sibylle , Roos Norbert , Krijnse Locker Jacomine
Published: September 2002

Vaccinia virus
(VV), a member of the poxvirus family, is unique among most other DNA viruses in that both transcription and DNA replication occur in the cytoplasm of the host cell. It was recently shown by electron microscopy (EM) that soon after viral DNA synthesis is initiated in HeLa cells, the replication sites become enwrapped by the membrane of the endoplasmic reticulum (ER). In the same study, a novel VV membrane protein, the E8R gene product, that may play a role in the ER wrapping process was identified (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001). In the present study, the gene product of E8R was characterized both biochemically and morphologically. We show that E8R is made predominantly early in infection but is packaged into the virion. On two-dimensional gel electrophoresis, the protein appeared as a single spot throughout the VV life cycle; however, in the assembled virion, the protein underwent several modifications which resulted in a change in its molecular weight and its isoelectric point. EM of labeled cryosections of infected HeLa cells showed that the protein localized to the ER and to membranes located on one side of the Golgi complex as early as 1 h postinfection. Late in infection, E8R was additionally associated with membranes of immature virions and with intracellular mature viruses. Although E8R is predominantly associated with membranes, we show that the protein is associated with viral cores; the protein is present in cores made with NP-40-dithiothreitol as well as in incoming cores, the result of the viral entry process, early in infection. Finally, we show that E8R can be phosphorylated in vitro by the viral kinase F10L. It is able to bind DNA in vitro, and this binding may be modulated by phosphorylation by F10L. A putative role of the E8R gene product throughout the VV life cycle is discussed.