bluuebunny/crossref_metadata_2025 huggingface dataset:
barS1, a Gene for Biosynthesis of a γ-Butyrolactone Autoregulator, a Microbial Signaling Molecule Eliciting Antibiotic Production inStreptomycesSpecies
Authors: Shikura Noriyasu , Yamamura Junji , Nihira Takuya
Published: October 2002

FromStreptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M1and S is tightly regulated by a low-molecular-weightStreptomyceshormone called virginiae butanolide (VB), which is a member of the γ-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression inEscherichia coliand by gene disruption inS. virginiae. ThebarS1gene (a 774-bp open reading frame encoding a 257-amino-acid protein [Mr, 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene,barA. The deduced BarS1 protein is weakly homologous to β-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In theΔbarS1mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by theΔbarS1mutant was fully recovered by the external addition of VB to the culture, which indicates that thebarS1gene is essential in the biosynthesis of the autoregulator VBs inS. virginiaeand that the failure of virginiamycin production was a result of the loss of VB production.
R391: a Conjugative Integrating Mosaic Comprised of Phage, Plasmid, and Transposon Elements
Authors: Böltner Dietmar , MacMahon Claire , Pembroke J. Tony , Strike Peter , Osborn A. Mark
Published: October 2002

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South AfricanProvidencia rettgeristrain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element fromVibrio choleraeO139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences fromSalmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.
Issues Associated with and Recommendations for Using PCR To Detect Outbreaks of Pertussis
Authors: Lievano Fabio A. , Reynolds Meredith A. , Waring Alfred L. , Ackelsberg Joel , Bisgard Kristine M. , Sanden Gary N. , Guris Dalya , Golaz Anne , Bopp Dianna J. , Limberger Ronald J. , Smith Perry F.
Published: October 2002

Two outbreaks of respiratory tract illness associated with prolonged cough occurring in 1998 and 1999 in New York State were investigated. A PCR test for
Bordetella pertussis
was primarily used by a private laboratory to confirm 680 pertussis cases. Several clinical specimens had positive culture results for
B. pertussis
during both outbreaks, which confirmed that
B. pertussis
was circulating during the outbreaks. However, testing by the New York State Department of Health reference laboratory suggested that some of the PCR results may have been falsely positive. In addition, features of the outbreak that suggested that
B. pertussis
may not have been the primary agent of infection included a low attack rate among incompletely vaccinated children and a significant amount of illness among patients testing PCR negative for
B. pertussis
. These investigations highlight the importance of appropriate clinical laboratory quality assurance programs, of the limitations of the PCR test, and of interpreting laboratory results in context of clinical disease.
Pharmacoepidemiological Analysis of Provincial Differences between Consumption of Macrolides and Rates of Erythromycin Resistance among
Streptococcus pyogenes
Isolates in Spain
Authors: García-Rey C. , Aguilar L. , Baquero F. , Casal J. , Martín J. E.
Published: October 2002

The M phenotype is by far the most common mechanism of erythromycin resistance among
Streptococcus pyogenes
isolates in Spain. A geographic analysis of the relationship between within-country differences in the prevalence of M-type resistance to erythromycin in
S. pyogenes
and the level of consumption of 14- and 15-membered macrolides within different provinces was carried out. From 1998 to 1999, a nationwide multicenter surveillance study yielded 2,039 consecutive pharyngeal isolates of
S. pyogenes
. Data on antibiotic consumption for the same period were gathered from IMS Health, and the corresponding daily defined doses per 1,000 inhabitants per day were calculated according to the Anatomic Therapeutic Classification index. Macrolide use was subdivided into dosages given three times a day (TID), twice a day (BID), or once a day (OD). Spearman nonparametric correlation coefficients (
R
) were calculated, and variables proving to be significantly associated (
P
< 0.1) were introduced into a linear regression model. The total consumption of macrolides presented a significant correlation with the prevalence of resistance (
R
= 0.527;
P
= 0.032). Neither TID nor BID macrolide consumption showed significant correlations. Only OD consumption had a significance below 0.1. These data are consistent with the hypothesis that only the total consumption of macrolides influences the local rates of M-type erythromycin resistance in
S. pyogenes,
and subgroups of macrolides seem to have an additive rather than a selective effect by contributing to increasing the final amount of macrolides used. Local variations in total consumption were associated only with BID consumption (
R
= 0.849;
P
= 0.004). The simple linear regression with total macrolide consumption showed a considerable determination coefficient (
R
2
= 0.678;
P
= 0.006). The model explains up to 68% of the measured variation and is clearly better as a predictor of the prevalence of resistance than the mere mean is. By solving the regression equation, the resultant value of 2.2 defined doses per 1,000 inhabitants per day fits with the existence of a critical threshold of selective pressure.
External Quality Assessment Program for Qualitative and Quantitative Detection of Hepatitis C Virus RNA in Diagnostic Virology
Authors: Schirm Jurjen , van Loon Anton M. , Valentine-Thon Elizabeth , Klapper Paul E. , Reid Jim , Cleator Graham M.
Published: October 2002

To assess the performance of laboratories in detecting and quantifying hepatitis C virus (HCV) RNA levels in HCV-infected patients, we distributed two proficiency panels for qualitative and quantitative HCV RNA testing. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each panel consisted of two negative samples and six positive samples, with HCV RNA target levels from 200 to 500,000 copies/ml. Panel 1 had four samples with at least 50,000 copies/ml, and panel 2 had two samples with at least 50,000 copies/ml. Fifty-seven laboratories submitted 45 qualitative and 35 quantitative data sets on panel 1, and 81 laboratories submitted 75 qualitative and 48 quantitative data sets on panel 2. In both panels, about two-thirds of the qualitative data sets and >90% of the quantitative data sets were obtained with commercial assays. With each panel, two data sets gave one false-positive result, corresponding to false-positivity rates of 1.3% and 0.8% for panel 1 and panel 2, respectively. Samples containing at least 50,000 copies/ml were found positive in 97% and 99% of the cases with panel 1 and panel 2, respectively. In contrast, the positive samples containing ≤5,000 copies/ml were reported positive in only 71% and 77% of the cases with panel 1 and panel 2, respectively. Adequate or better scores on qualitative results (all results correct or only the low-positive samples missed) were obtained in 84% (panel 1) and 80% (panel 2) of the data sets. In the analysis of quantitative results, 60% (panel 1) and 73% (panel 2) of the data sets obtained an adequate or better score (≥80% of the positive results within the range of the geometric mean ± 0.5 log
10
). Our results indicate that considerable improvements in molecular detection and quantitation of HCV have been achieved, particularly through the use of commercial assays. However, the lowest detection levels of many assays are still too high, and further standardization is still needed. Finally, this study underlines the importance of proficiency panels for monitoring the quality of diagnostic laboratories.
Analysis of Sequences and Loci of
p44
Homologs Expressed by
Anaplasma phagocytophila
in Acutely Infected Patients
Authors: Lin Quan , Zhi Ning , Ohashi Norio , Horowitz Harold W. , Aguero-Rosenfeld Maria E. , Raffalli John , Wormser Gary P. , Rikihisa Yasuko
Published: October 2002

Anaplasma phagocytophila
is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of
A. phagocytophila
are encoded by a
p44
multigene family. In the present study, expression profiles of
p44
genes in the blood of acutely infected patients in the year 2000 were characterized. A single
p44
gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different
p44
genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several
p44
genes were identical among the isolates from the three patients and a 1995
A. phagocytophila
isolate.
A. phagocytophila
was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995
A. phagocytophila
isolates with probes specific to the most dominant
p44
transcript in each patient showed that the
p44
loci in the
A. phagocytophila
genome were conserved. Analysis of the predicted amino acid sequences of 43 different
p44
genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of
p44
are not random but are restricted. Furthermore, several
p44
genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.
Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii
Authors: Larsen Hans Henrik , Kovacs Joseph A. , Stock Frida , Vestereng Vibeke H. , Lundgren Bettina , Fischer Steven H. , Gill Vee J.
Published: October 2002

A method for reliable quantification ofPneumocystis cariniiin research models ofP. cariniipneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detectingP. cariniif. sp.carinii, the subspecies ofP. cariniicommonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r= 0.99) over 6 log values for standards containing ≥5 copies/tube. Application of the assay to a series of 10-fold dilutions ofP. cariniiorganisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r= 0.99). The assay was applied to a recently reported in vitro axenic cultivation system forP. cariniiand confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease inP. cariniiDNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay forP. cariniif. sp.cariniihas been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.
Large-Scale Screening of Nasal Swabs for
Bacillus anthracis
: Descriptive Summary and Discussion of the National Institutes of Health's Experience
Authors: Kiratisin Pattarachai , Fukuda Caroline D. , Wong Alexandra , Stock Frida , Preuss Jeanne C. , Ediger Laura , Brahmbhatt Trupti N. , Fischer Steven H. , Fedorko Daniel P. , Witebsky Frank G. , Gill Vee J.
Published: October 2002

In October 2001, a letter containing a large number of anthrax spores was sent through the Brentwood post office in Washington, D.C., to a United States Senate office on Capitol Hill, resulting in contamination in both places. Several thousand people who worked at these sites were screened for spore exposure by collecting nasal swab samples. We describe here a screening protocol which we, as a level A laboratory, used on very short notice to process a large number of specimens (3,936 swabs) in order to report preliminary results as quickly as possible. Six isolates from our screening met preliminary criteria for
Bacillus anthracis
identification and were referred for definitive testing. Although none of the isolates was later confirmed to be
B. anthracis
, we studied these isolates further to define their biochemical characteristics and 16S rRNA sequences. Four of the six isolates were identified as
Bacillus megaterium
, one was identified as
Bacillus cereus
, and one was an unidentifiable
Bacillus
sp. Our results suggest that large-scale nasal-swab screening for potential exposure to anthrax spores, particularly if not done immediately postexposure, may not be very effective for detecting
B. anthracis
but may detect a number of
Bacillus
spp. that are phenotypically very similar to
B. anthracis
.
Presence of Integron-Associated Resistance in the Community Is Widespread and Contributes to Multidrug Resistance in the Hospital
Authors: Leverstein-van Hall M. A. , Paauw A. , Box A. T. A. , Blok H. E. M. , Verhoef J. , Fluit A. C.
Published: October 2002

Integrons are strongly associated with the multidrug resistance seen in gram-negative bacilli in the hospital environment. No data, however, are available on their prevalence in the community. This study is the first to show that integrons are widespread in
Enterobacteriaceae
in the community and that integron-associated resistance genes in the community constitute a substantial reservoir for multidrug resistance in the hospital.
Hepatitis D Virus Genotypes in Intravenous Drug Users in Taiwan: Decreasing Prevalence and Lack of Correlation with Hepatitis B Virus Genotypes
Authors: Kao Jia-Horng , Chen Pei-Jer , Lai Ming-Yang , Chen Ding-Shinn
Published: October 2002

Of 368 hepatitis B virus (HBV)-infected intravenous drug users, 144 (39%) were positive for antibody to hepatitis D virus (anti-HDV). Anti-HDV-positive HBV carriers had a lower rate of positivity for HBV DNA than did anti-HDV-negative carriers (52 versus 73%, respectively). From 1986 to 1997, the average rate of decrease in the prevalence of HDV infection in this population was 4.7%/year.
Molecular Characterization of Preintegration Latency in Human Immunodeficiency Virus Type 1 Infection
Authors: Pierson Theodore C. , Zhou Yan , Kieffer Tara L. , Ruff Christian T. , Buck Christopher , Siliciano Robert F.
Published: October 2002

Most current evidence suggests that the infection of resting CD4+T cells by human immunodeficiency virus type 1 (HIV-1) is not productive due to partial or complete blocks in the viral life cycle at steps prior to integration of the viral genome into the host cell chromosome. However, stimulation of an infected resting T cell by antigen, cytokines, or microenvironmental factors can overcome these blocks and allow for the production of progeny virions. In this study, we sought to understand the structure and fate of the virus in unstimulated resting CD4+T cells. Using a novel linker-mediated PCR assay designed to detect and characterize linear unintegrated forms of the HIV-1 genome, we demonstrate that reverse transcription can proceed to completion following the infection of resting T cells, generating the substrate for the retroviral integration reaction. However, reverse transcription in resting T cells is far slower than in activated T cells, requiring 2 to 3 days to complete. The delay in completing reverse transcription may make the viral DNA genome more susceptible to competing decay processes. To explore the relationship between the formation of the linear viral genome and the stability of the preintegration state, we employed a recombinant HIV-1 virus expressing the enhanced green fluorescent protein to measure the rate at which HIV-1 decays in the preintegration state. Our results demonstrate that the preintegration state is labile and decays rapidly (half-life = 1 day) following the entry of HIV-1 into a resting T cell, with significant decay occurring during the slow process of reverse transcription.
Ebola Virus VP30-Mediated Transcription Is Regulated by RNA Secondary Structure Formation
Authors: Weik Michael , Modrof Jens , Klenk Hans-Dieter , Becker Stephan , Mühlberger Elke
Published: October 2002

The nucleocapsid protein VP30 of
Ebola virus
(EBOV), a member of the
Filovirus
family, is known to act as a transcription activator. By using a reconstituted minigenome system, the role of VP30 during transcription was investigated. We could show that VP30-mediated transcription activation is dependent on formation of a stem-loop structure at the first gene start site. Destruction of this secondary structure led to VP30-independent transcription. Analysis of the transcription products of bicistronic minigenomes with and without the ability to form the secondary structure at the first transcription start signal revealed that transcription initiation at the first gene start site is a prerequisite for transcription of the second gene, independent of the presence of VP30. When the transcription start signal of the second gene was exchanged with the transcription start signal of the first gene, transcription of the second gene also was regulated by VP30, indicating that the stem-loop structure of the first transcription start site acts autonomously and independently of its localization on the RNA genome. Our results suggest that VP30 regulates a very early step of EBOV transcription, most likely by inhibiting pausing of the transcription complex at the RNA structure of the first transcription start site.
Naturally Acquired Simian Varicella Virus Infection in African Green Monkeys
Authors: Mahalingam Ravi , Traina-Dorge Vicki , Wellish Mary , Smith John , Gilden Donald H.
Published: October 2002

Simian varicella virus (SVV) infection of primates shares clinical, pathological, immunological, and virological features with varicella-zoster virus infection of humans. Natural varicella infection was simulated by exposing four SVV-seronegative monkeys to monkeys inoculated intratracheally with SVV, in which viral DNA and RNA persist in multiple tissues for more than 1 year (T. M. White, R. Mahalingam, V. Traina-Dorge, and D. H. Gilden, J. Neurovirol.
8:
191-205, 2002). The four naturally exposed monkeys developed mild varicella 10 to 14 days later, and skin scrapings taken at the time of the rash contained SVV DNA. Analysis of multiple ganglia, liver, and lung tissues from the four naturally exposed monkeys sacrificed 6 to 8 weeks after resolution of the rash revealed SVV DNA in ganglia at multiple levels of the neuraxis but not in the lung or liver tissue of any of the four monkeys. This animal model provides an experimental system to gain information about varicella latency with direct relevance to the human disease.
mRNA Degradation by the Virion Host Shutoff (Vhs) Protein of Herpes Simplex Virus: Genetic and Biochemical Evidence that Vhs Is a Nuclease
Authors: Everly, David N. , Feng Pinghui , Mian I. Saira , Read G. Sullivan
Published: October 2002

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating the decay of all mRNAs, it helps redirect the cell from host to viral gene expression and facilitates the sequential expression of different classes of viral genes. While it is clear that Vhs induces mRNA degradation, it is uncertain whether it is itself an RNase or somehow activates a cellular enzyme. This question was addressed by using a combination of genetic and biochemical approaches. The Vhs homologues of alphaherpesviruses share sequence similarities with a family of mammalian, yeast, bacterial, and phage nucleases. To test the functional significance of these similarities, Vhs was mutated to alter residues corresponding to amino acids known to be critical to the nuclease activity of cellular homologues. In every instance, mutations that inactivated the nuclease activity of cellular homologues also abolished Vhs activity. Recent experiments showed that Vhs interacts with the cellular translation initiation factor eIF4H. In this study, the coexpression of Vhs and a glutathioneS-transferase (GST)-eIF4H fusion protein in bacteria resulted in the formation of a complex of the proteins. The wild-type Vhs/GST-eIF4H complex was isolated and shown to have RNase activity. In contrast, Vhs mutations that altered key residues in the nuclease motif abolished the nuclease activity of the recombinant Vhs/GST-eIF4H complex. The results provide genetic and biochemical evidence that Vhs is an RNase, either alone or as a complex with eIF4H.
Clustering Patterns of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1 (HIV-1) Proteins Reveal Imprints of Immune Evasion on HIV-1 Global Variation
Authors: Yusim Karina , Kesmir Can , Gaschen Brian , Addo Marylyn M. , Altfeld Marcus , Brunak Søren , Chigaev Alexandre , Detours Vincent , Korber Bette T.
Published: October 2002

The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequences reveal that defined epitopes tend to cluster. Here we integrate the global sequence and immunology databases to systematically explore the relationship between HIV-1 amino acid sequences and CTL epitope distributions. CTL responses to five HIV-1 proteins, Gag p17, Gag p24, reverse transcriptase (RT), Env, and Nef, have been particularly well characterized in the literature to date. Through comparing CTL epitope distributions in these five proteins to global protein sequence alignments, we identified distinct characteristics of HIV amino acid sequences that correlate with CTL epitope localization. First, experimentally defined HIV CTL epitopes are concentrated in relatively conserved regions. Second, the highly variable regions that lack epitopes bear cumulative evidence of past immune escape that may make them relatively refractive to CTLs: a paucity of predicted proteasome processing sites and an enrichment for amino acids that do not serve as C-terminal anchor residues. Finally, CTL epitopes are more highly concentrated in alpha-helical regions of proteins. Based on amino acid sequence characteristics, in a blinded fashion, we predicted regions in HIV regulatory and accessory proteins that would be likely to contain CTL epitopes; these predictions were then validated by comparison to new sets of experimentally defined epitopes in HIV-1 Rev, Tat, Vif, and Vpr.
Generation of Neutralizing Activity against Human Immunodeficiency Virus Type 1 in Serum by Antibody Gene Transfer
Authors: Lewis Anne D. , Chen Ruju , Montefiori David C. , Johnson Philip R. , Clark K. Reed
Published: October 2002

Although several human immunodeficiency virus (HIV) vaccine approaches have elicited meaningful antigen-specific T-cell responses in animal models, no single vaccine candidate has engendered antibodies that broadly neutralize primary isolates of HIV type 1 (HIV-1). Thus, there remains a significant gap in the design of HIV vaccines. To address this issue, we exploited the existence of rare human monoclonal antibodies that have been isolated from HIV-infected individuals. Such antibodies neutralize a wide array of HIV-1 field isolates and have been shown to be effective in vivo. However, practical considerations preclude the use of antibody preparations as a prophylactic passive immunization strategy in large populations. Our concept calls for an antibody gene of choice to be transferred to muscle where the antibody molecule is synthesized and distributed to the circulatory system. In these experiments, we used a recombinant adeno-associated virus (rAAV) vector to deliver the gene for the human antibody IgG1b12 to mouse muscle. Significant levels of HIV-neutralizing activity were found in the sera of mice for over 6 months after a single intramuscular administration of the rAAV vector. This approach allows for predetermination of antibody affinity and specificity prior to “immunization” and avoids the need for an active humoral immune response against the HIV envelope protein.
Intranasal Vaccination with a Recombinant Vesicular Stomatitis Virus Expressing Cottontail Rabbit Papillomavirus L1 Protein Provides Complete Protection against Papillomavirus-Induced Disease
Authors: Reuter Jon D. , Vivas-Gonzalez Beatriz E. , Gomez Daniel , Wilson Jean H. , Brandsma Janet L. , Greenstone Heather L. , Rose John K. , Roberts Anjeanette
Published: October 2002

Immunizations with live recombinant vesicular stomatitis viruses (rVSV) expressing foreign viral proteins have successfully protected animals from challenges with several heterologous viruses. We developed an rVSV expressing the major capsid protein (L1) of cottontail rabbit papillomavirus (CRPV) and tested the efficacy of protection following CRPV challenge. An rVSV expressing L1 of CRPV (VSV-L1) was characterized for the protective ability afforded by intranasal, intradermal, or intramuscular vaccination in rabbits subsequently challenged with CRPV. Protein expression of L1 in VSV-L1 was confirmed by radioimmunoprecipitation assays. Nuclear localization of L1 was demonstrated by indirect immunofluorescence assays. Immunized rabbits elicited significant VSV neutralization and VLP-L1 enzyme-linked immunosorbent assay titers. VSV-L1 vaccination was not associated with weight loss or any other adverse clinical signs in the rabbit model. VSV shedding in nasal secretions occurred in some rabbits, peaking at 4 to 6 days after intranasal vaccination, with no further shedding after day 6. Specific humoral immunity to the L1 protein was consistently seen after a single VSV-L1 vaccination when administered through an intradermal or intramuscular route or after a boost via the intranasal route. Rabbits were completely protected from CRPV-induced papillomas after VSV-L1 vaccination and boost given intranasally or intramuscularly. Vaccination with VSV-L1 is a novel approach to prevent papillomavirus-induced disease and demonstrates a potential strategy for developing a human papillomavirus vaccine that can be given without injection.
Molecular Analysis of Three Ljungan Virus Isolates Reveals a New, Close-to-Root Lineage of the
Picornaviridae
with a Cluster of Two Unrelated 2A Proteins
Authors: Johansson Susanne , Niklasson Bo , Maizel Jacob , Gorbalenya Alexander E. , Lindberg A. Michael
Published: October 2002

Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles (
Clethrionomys glareolus
). In the present study, it is revealed through comparative sequence analysis that three newly determined Swedish LV genomes are closely related and possess a deviant picornavirus-like organization: 5′ untranslated region-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3′ untranslated region. The LV genomes and the polyproteins encoded by them exhibit several exceptional features, such as the absence of a predicted maturation cleavage of VP0, a conserved sequence determinant in VP0 that is typically found in VP1 of other picornaviruses, and a cluster of two unrelated 2A proteins. The 2A1 protein is related to the 2A protein of cardio-, erbo-, tescho-, and aphthoviruses, and the 2A2 protein is related to the 2A protein of parechoviruses, kobuviruses, and avian encephalomyelitis virus. The unprecedented association of two structurally different 2A proteins is a feature never previously observed among picornaviruses and implies that their functions are not mutually exclusive. Secondary polyprotein processing of the LV polyprotein is mediated by proteinase 3C (3C
pro
) possessing canonical affinity to Glu and Gln at the P1 position and small amino acid residues at the P1′ position. In addition, LV 3C
pro
appears to have unique substrate specificity to Asn, Gln, and Asp and to bulky hydrophobic residues at the P2 and P4 positions, respectively. Phylogenetic analysis suggests that LVs form a separate division, which, together with the
Parechovirus
genus, has branched off the picornavirus tree most closely to its root. The presence of two 2A proteins indicates that some contemporary picornaviruses with a single 2A may have evolved from the ancestral multi-2A picornavirus.
Ultrastructural Localization of the Herpes Simplex Virus Type 1 U
L
31, U
L
34, and U
S
3 Proteins Suggests Specific Roles in Primary Envelopment and Egress of Nucleocapsids
Authors: Reynolds Ashley E. , Wills Elizabeth G. , Roller Richard J. , Ryckman Brent J. , Baines Joel D.
Published: October 2002

The wild-type U
L
31, U
L
34, and U
S
3 proteins localized on nuclear membranes and perinuclear virions; the U
S
3 protein was also on cytoplasmic membranes and extranuclear virions. The U
L
31 and U
L
34 proteins were not detected in extracellular virions. U
S
3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the U
S
3 protein plays an important, but nonessential, role in the egress pathway.
Quantitative Expression and Virus Transmission Analysis of DC-SIGN on Monocyte-Derived Dendritic Cells
Authors: Baribaud Frédéric , Pöhlmann Stefan , Leslie George , Mortari Frank , Doms Robert W.
Published: October 2002

The C-type lectins DC-SIGN and DC-SIGNR efficiently bind human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains and can transmit bound virus to adjacent CD4-positive cells. DC-SIGN also binds efficiently to the Ebola virus glycoprotein, enhancing Ebola virus infection. DC-SIGN is thought to be responsible for the ability of dendritic cells (DCs) to capture HIV and transmit it to T cells, thus promoting HIV dissemination in vitro and perhaps in vivo as well. To investigate DC-SIGN function and expression levels on DCs, we characterized a panel of monoclonal antibodies (MAbs) directed against the carbohydrate recognition domain of DC-SIGN. Using quantitative fluorescence-activated cell sorter technology, we found that DC-SIGN is highly expressed on immature monocyte-derived DCs, with at least 100,000 copies and often in excess of 250,000 copies per DC. There was modest variation (three- to fourfold) in DC-SIGN expression levels between individuals and between DCs isolated from the same individual at different times. Several MAbs efficiently blocked virus binding to cell lines expressing human or rhesus DC-SIGN, preventing HIV and SIV transmission. Interactions with Ebola virus pseudotypes were also blocked efficiently. Despite their ability to block virus-DC-SIGN interactions on cell lines, these antibodies only inhibited transmission of virus from DCs by approximately 50% or less. These results indicate that factors other than DC-SIGN may play important roles in the ability of DCs to capture and transmit HIV.
Persisting Murine Cytomegalovirus Can Reactivate and Has Unique Transcriptional Activity in Ocular Tissue
Authors: Kercher Lisa , Mitchell Bradley M.
Published: October 2002

Cytomegalovirus (CMV) retinitis is an important ocular complication in human immunodeficiency virus-infected individuals and the leading cause of blindness in those not undergoing highly active antiretroviral therapy. Murine CMV (MCMV) infection of mice has been shown to be a useful small-animal model for the study of CMV pathogenesis in the eye. The purpose of this study was to evaluate CMV persistence in ocular tissue and to determine the potential for reactivation. Following subretinal inoculation of immunocompetent BALB/c mice, tissues were tested for infectious virus by plaque assay and for the presence of viral DNA and RNA by PCR. The latent phase of the infection in mouse tissues was analyzed by plaque assay, PCR, and explantation cocultivation in both immunocompetent and cyclophosphamide-treated mice. The acute phase of the infection was resolved by 2 to 3 weeks postinfection, while viral DNA persisted beyond 12 months. Immediate-early 1 transcripts were detected in 100% of the ocular samples tested, and glycoprotein H transcripts were detected in 86% of the samples, but no difference in viral DNA or RNA levels between immunocompetent and immunosuppressed animals was measured. Irrespective of immune status, no in vivo reactivation was detected; however, reactivated virus was observed in 76 to 82% of the eyes following explantation onto a permissive cell layer. The transcriptional activity and relatively high frequency of explantation-induced reactivation in both immunocompetent and immunosuppressed mice suggest that control of MCMV latency in ocular tissue might involve other regulatory events that are not entirely dependent on intact specific immunity.
Induction of Immune Responses in Mice and Monkeys to Ebola Virus after Immunization with Liposome-Encapsulated Irradiated Ebola Virus: Protection in Mice Requires CD4
+
T Cells
Authors: Rao Mangala , Bray Mike , Alving Carl R. , Jahrling Peter , Matyas Gary R.
Published: October 2002

Ebola Zaire virus (EBO-Z) causes severe hemorrhagic fever in humans, with a high mortality rate. It is thought that a vaccine against EBO-Z may have to induce both humoral and cell-mediated immune responses to successfully confer protection. Because it is known that liposome-encapsulated antigens induce both antibody and cellular responses, we evaluated the protective efficacy of liposome-encapsulated irradiated EBO-Z [L(EV)], which contains all of the native EBO-Z proteins. In a series of experiments, mice immunized intravenously with L(EV) were completely protected (94/94 mice) against illness and death when they were challenged with a uniformly lethal mouse-adapted variant of EBO-Z. In contrast, only 55% of mice immunized intravenously with nonencapsulated irradiated virus (EV) survived challenge, and all became ill. Treatment with anti-CD4 antibodies before or during immunization with L(EV) eliminated protection, while treatment with anti-CD8 antibodies had no effect, thus indicating a requirement for CD4
+
T lymphocytes for successful immunization. On the other hand, treatment with either anti-CD4 or anti-CD8 antibodies after immunization did not abolish the protection. After immunization with L(EV), antigen-specific gamma interferon (IFNγ)-secreting CD4
+
T lymphocytes were induced as analyzed by enzyme-linked immunospot assay. Anti-CD4 monoclonal antibody treatment abolished IFNγ production (80 to 90% inhibition compared to that for untreated mice). Mice immunized with L(EV), but not EV, developed cytotoxic T lymphocytes specific to two peptides (amino acids [aa] 161 to 169 and aa 231 to 239) present in the amino-terminal end of the EBO-Z surface glycoprotein. Because of the highly successful results in the mouse model, L(EV) was also tested in three cynomolgus monkeys. Although immunization of the monkeys with L(EV)-induced virus-neutralizing antibodies against EBO-Z caused a slight delay in the onset of illness, it did not prevent death.
Subversion of Cell Signaling Pathways by Hepatitis C Virus Nonstructural 5A Protein via Interaction with Grb2 and P85 Phosphatidylinositol 3-Kinase
Authors: He Yupeng , Nakao Haruhisa , Tan Seng-Lai , Polyak Stephen J. , Neddermann Petra , Vijaysri Sangeetha , Jacobs Bertram L. , Katze Michael G.
Published: October 2002

Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.
Cleavage at the Furin Consensus Sequence RAR/KR
109
and Presence of the Intervening Peptide of the Respiratory Syncytial Virus Fusion Protein Are Dispensable for Virus Replication in Cell Culture
Authors: Zimmer Gert , Conzelmann Karl-Klaus , Herrler Georg
Published: October 2002

Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR
136
) and FCS-2 (RAR/KR
109
). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR
109
, RANN
109
or SANN
109
, respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR
109
or the RANN
109
motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN
109
mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.
Roles for the Cytoplasmic Tails of the Fusion and Hemagglutinin-Neuraminidase Proteins in Budding of the Paramyxovirus Simian Virus 5
Authors: Waning David L. , Schmitt Anthony P. , Leser George P. , Lamb Robert A.
Published: October 2002

The efficient release of many enveloped viruses from cells involves the coalescence of viral components at sites of budding on the plasma membrane of infected cells. This coalescence is believed to require interactions between the cytoplasmic tails of surface glycoproteins and the matrix (M) protein. For the paramyxovirus simian virus 5 (SV5), the cytoplasmic tail of the hemagglutinin-neuraminidase (HN) protein has been shown previously to be important for normal virus budding. To investigate a role for the cytoplasmic tail of the fusion (F) protein in virus assembly and budding, we generated a series of F cytoplasmic tail-truncated recombinant viruses. Analysis of these viruses in tissue culture indicated that the cytoplasmic tail of the F protein was dispensable for normal virus replication and budding. To investigate further the requirements for assembly and budding of SV5, we generated two double-mutant recombinant viruses that lack 8 amino acids of the predicted 17-amino-acid HN protein cytoplasmic tail in combination with truncation of either 10 or 18 amino acids from the predicted 20-amino-acid F protein cytoplasmic tail. Both of the double mutant recombinant viruses displayed a replication defect in tissue culture and a budding defect, the extent of which was dependant on the length of the remaining F cytoplasmic tail. Taken together, this work and our earlier data on virus-like particle formation (A. P. Schmitt, G. P. Leser, D. L. Waning, and R. A. Lamb, J. Virol. 76:3953-3964, 2002) suggest a redundant role for the cytoplasmic tails of the HN and F proteins in virus assembly and budding.
Interaction of Hepatitis C Virus-Like Particles and Cells: a Model System for Studying Viral Binding and Entry
Authors: Triyatni Miriam , Saunier Bertrand , Maruvada Padma , Davis Anthony R. , Ulianich Luca , Heller Theo , Patel Arvind , Kohn Leonard D. , Liang T. Jake
Published: October 2002

Hepatitis C virus-like particles (HCV-LPs) containing the structural proteins of HCV H77 strain (1a genotype) was used as a model for HCV virion to study virus-cell interaction. HCV-LPs showed a buoyant density of 1.17 to 1.22 g/cm
3
in a sucrose gradient and formed double-shelled particles 35 to 49 nm in diameter. Flow cytometry analysis by an indirect method (detection with anti-E2 antibody) and a direct method (use of dye-labeled HCV-LPs) showed that HCV-LPs binds to several human hepatic (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cell (Molt-4) lines. HCV-LPs binding to cells occurred in a dose- and calcium-dependent manner and was not mediated by CD81. Scatchard plot analysis suggests the presence of two binding sites for HCV-LPs with high (

K
d

∼1 μg/ml) and low (

K
d

∼50 to 60 μg/ml) affinities of binding. Anti-E1 and -E2 antibodies inhibited HCV-LPs binding to cells. While preincubation of HCV-LPs with very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), or high-density lipoprotein (HDL) blocked its binding to cells, preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Confocal microscopy analysis showed that, after binding to cells, dye-labeled HCV-LPs were internalized into the cytoplasm. This process could be inhibited with anti-E1 or anti-E2 antibodies, suggesting that E1 and E2 proteins mediate HCV-LPs binding and, subsequently, their entry into cells. Altogether, our results indicate that HCV-LPs can be used to further characterize the mechanisms involved in the early steps of HCV infection.
Signals That Dictate Nuclear, Nucleolar, and Cytoplasmic Shuttling of the γ
1
34.5 Protein of Herpes Simplex Virus Type 1
Authors: Cheng Guofeng , Brett Marie-Elena , He Bin
Published: October 2002

The γ
1
34.5 protein of herpes simplex virus type 1 (HSV-1) is required for viral neurovirulence in vivo. In infected cells, this viral protein prevents the shutoff of protein synthesis mediated by double-stranded-RNA-dependent protein kinase PKR. This is accomplished by recruiting protein phosphatase 1 to dephosphorylate the α subunit of translation initiation factor eIF-2 (eIF-2α). Moreover, the γ
1
34.5 protein is implicated in viral egress and interacts with proliferating cell nuclear antigen. In this report, we show that the γ
1
34.5 protein encoded by HSV-1(F) is distributed in the nucleus, nucleolus, and cytoplasm in transfected or superinfected cells. Deletion analysis revealed that the Arg-rich cluster from amino acids 1 to 16 in the γ
1
34.5 protein functions as a nucleolar localization signal. The region from amino acids 208 to 236, containing a bipartite basic amino acid cluster, is able to mediate nuclear localization. R
215
A and R
216
A substitutions in the bipartite motif disrupt this activity. Intriguingly, leptomycin B, an inhibitor of nuclear export, blocks the cytoplasmic accumulation of the γ
1
34.5 protein. L
134
A and L
136
A substitutions in the leucine-rich motif completely excluded the γ
1
34.5 protein from the cytoplasm. These results suggest that the γ
1
34.5 protein continuously shuttles between the nucleus, nucleolus, and cytoplasm, which may be a requirement for the different activities of the γ
1
34.5 protein in virus-infected cells.
Interferon Regulatory Factor 3 Is a Cellular Partner of Rotavirus NSP1
Authors: Graff Joel W. , Mitzel Dana N. , Weisend Carla M. , Flenniken Michelle L. , Hardy Michele E.
Published: October 2002

The rotavirus nonstructural protein NSP1 is the least conserved protein in the rotavirus genome, and its function in the replication cycle is not known. We employed NSP1 as bait in the yeast two-hybrid interaction trap to identify candidate cellular partners of NSP1 that may provide clues to its function. Interferon regulatory factor 3 (IRF-3) was identified as an NSP1 interactor. NSP1 synthesized in rotavirus-infected cells bound IRF-3 in a glutathioneS-transferase pull-down assay, indicating that the interaction was not unique to the two-hybrid system. NSP1 of murine rotavirus strain EW also interacted with IRF-3. NSP1 deletion and point mutants were constructed to map domains important in the interaction between NSP1 and IRF-3. The data suggest that a binding domain resides in the C terminus of NSP1 and that the N-terminal conserved zinc finger is important but not sufficient to mediate binding to IRF-3. We predict that a role for NSP1 in rotavirus-infected cells is to inhibit activation of IRF-3 and diminish the cellular interferon response.