bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of PCR-Restriction Profile Analysis and IS
2404
Restriction Fragment Length Polymorphism and Amplified Fragment Length Polymorphism Fingerprinting for Identification and Typing of
Mycobacterium ulcerans
and
M. marinum
Authors: Chemlal K. , Huys G. , Fonteyne P.-A. , Vincent V. , Lopez A. G. , Rigouts L. , Swings J. , Meyers W. M. , Portaels F.
Published: July 2002

Mycobacterium ulcerans
and
M. marinum
are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS
2404
restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of
M. ulcerans
(
n
= 29) and
M. marinum
(
n
= 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3′ end region of 16S rRNA, which differentiated
M. ulcerans
into three types; however, the technique could not distinguish
M. marinum
from
M. ulcerans
isolates originating from South America and Southeast Asia. RFLP based on IS
2404
produced six
M. ulcerans
types related to six geographic regions and did not produce any band with
M. marinum
, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270–273, 2001). AFLP analysis resulted in profiles which grouped
M. ulcerans
and
M. marinum
into two separate clusters. The numerical analysis also revealed subgroups among the
M. marinum
and
M. ulcerans
isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African
M. ulcerans
type from other geographical types but is unsuitable for interspecific differentiation of
M. marinum
and
M. ulcerans
. In comparison, whole- genome techniques such as IS
2404
-RFLP and AFLP appear to be far more useful in discriminating between
M. marinum
and
M. ulcerans
, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.
Selective Amplification of
tyv
(rfbE),
prt
(rfbS),
viaB
, and
fliC
Genes by Multiplex PCR for Identification of
Salmonella enterica
Serovars Typhi and Paratyphi A
Authors: Hirose Kenji , Itoh Ken-Ichiro , Nakajima Hiroshi , Kurazono Takayuki , Yamaguchi Masanori , Moriya Kazuo , Ezaki Takayuki , Kawamura Yoshiaki , Tamura Kazumichi , Watanabe Haruo
Published: July 2002

The PCR primers for O, H, and Vi antigen genes,
tyv
(rfbE),
prt
(
rfbS
),
fliC-d, fliC-a
, and
viaB
, were designed and used for the rapid identification of
Salmonella enterica
serovars Typhi and Paratyphi A with multiplex PCR. The results showed that all the clinical isolates examined of
Salmonella
serovars Typhi and Paratyphi A were accurately identified by this assay.
Rapidly Increasing Prevalence of Penicillin-Resistant
Streptococcus
pneumoniae
in Middle Tennessee: a 10-Year Clinical and Molecular Analysis
Authors: Tang Yi-Wei , Li Haijing , Griffin Jane P. , Haas David W. , D'Agata Erika M. C.
Published: July 2002

The clinical and molecular epidemiology of penicillin-resistant
Streptococcus
pneumoniae
and the diagnostic accuracy of a six-primer PCR assay in identifying penicillin resistance were analyzed by using clinical isolates recovered over a 10-year period in middle Tennessee. The prevalence of non-penicillin-susceptible
S. pneumoniae
isolates (MIC, ≥0.1 μg/ml) increased from 10% in 1990 to 70% in 1999 (
P
< 0.001). Among
S. pneumoniae
isolates for which the penicillin MIC was ≥2 μg/ml (highly penicillin-resistant
S. pneumoniae
[PRSP]), 23 and 5% were resistant to at least three and at least five other antimicrobial classes, respectively. Pulsed-field gel electrophoresis identified 13 unique strain types, with type B accounting for 33% of PRSP isolates. The sensitivity, specificity, and negative and positive predictive values of the PCR assay in detecting PRSP were 99, 100, 99, and 100%, respectively. Penicillin resistance is rapidly increasing among
S. pneumoniae
isolates in Tennessee. The simultaneous detection of
S. pneumoniae
and high-level penicillin resistance can be accurately performed with the six-primer PCR assay.
Evaluation of Confirmatory Strategies for Detection of Type-Specific Antibodies against Herpes Simplex Virus Type 2
Authors: Eing Bodo Rolf , Lippelt Lars , Lorentzen Eva Ulla , Hafezi Wali , Schlumberger Wolfgang , Steinhagen Katja , Kühn Joachim Ewald
Published: July 2002

In this study, the optimal combination of three commercial glycoprotein G-2 (gG-2)-based herpes simplex virus type 2 (HSV-2) type-specific enzyme-linked immunosorbent assays (Euroimmun anti-HSV-2 immunoglobulin G [IgG] ELISA [Eu2], Gull HSV-2-specific IgG ELISA [Gu2], and Radim HSV-2 IgG ELISA [Ra2]) and one gG-2-based HSV-2-specific immunoblot (Euroimmun anti-HSV-1/HSV-2 gG Western blot [EuW]) was determined with regard to diagnostic performance and cost efficiency. Two hundred fifty serum samples were included in this study, 194 of which were from female prostitutes. When a formal primary “gold standard” was defined based on majority agreement of the commercial tests, with EuW being decisive in stand-off situations, the sensitivity and specificity of the assays in the samples from prostitutes were as follows: Eu2, 100 and 89.22%; Gu2, 94.44 and 96.08%; Ra2, 61.18 and 95.10%; and EuW, 98.90 and 100%. The most cost-effective confirmatory strategy in the samples from prostitutes was screening with Eu2, retesting positive and equivocal samples with Gu2, and resolving the remaining discordant results with EuW (estimated additional costs per sample, 79.02%; sensitivity, 100%; positive predictive value, 96.81%). Applying a self-developed gG-2-independent assay to the discordant and concordant negative samples in the samples from prostitutes suggested that the primary gold standard may have missed six HSV-2-positive samples. In conclusion, confirmatory strategies based on commercial gG-2-dependent seroassays result in an increase in the specificity of HSV-2-specific serology. However, further improvement of the sensitivity of current HSV-2-specific serology may require the additional exploitation of the gG-2-independent type-specific antibody response.
Evaluation of Automated BACTEC MGIT 960 System for Testing Susceptibility of
Mycobacterium tuberculosis
to Four Major Antituberculous Drugs: Comparison with the Radiometric BACTEC 460TB Method and the Agar Plate Method of Proportion
Authors: Tortoli Enrico , Benedetti Marta , Fontanelli Alessandra , Simonetti M. Tullia
Published: July 2002

We evaluated the performance of BACTEC MGIT 960 for automated testing of the susceptibility of 133 strains of
Mycobacterium tuberculosis
to streptomycin, isoniazid, rifampin, and ethambutol. The BACTEC MGIT 960 results were compared with those obtained with the radiometric BACTEC 460TB system, and when there was disagreement, the method of proportion on agar plates was used as a reference method. Strains resistant to the critical concentration of streptomycin, isoniazid, or ethambutol were also tested with a second, higher concentration. The overall agreement between the two systems was 96.7%, and the 18 discrepancies were resolved in favor of BACTEC 460TB in 11 cases and in favor of BACTEC MGIT 960 in 7, a difference which was not statistically significant. Apart from the assay's low specificity for ethambutol, which was low for the radiometric assay as well, good sensitivity and specificity values characterized BACTEC MGIT 960. The average time required for completion of the test was 2.5 days shorter with BACTEC 460TB. In conclusion, BACTEC MGIT 960 appears to be a suitable replacement for the radiometric method of antimicrobial susceptibility testing of
M. tuberculosis
. The problem of frequent contamination of BACTEC MGIT 960 tests needs to be quickly resolved; in fact, 14 strains had to be reprocessed because of contamination.
Spread of Drug-Resistant
Mycobacterium tuberculosis
Strains of the Beijing Genotype in the Archangel Oblast, Russia
Authors: Toungoussova Olga S. , Sandven Per , Mariandyshev Andrey O. , Nizovtseva Nina I. , Bjune Gunnar , Caugant Dominique A.
Published: July 2002

A collection of 119 strains of
Mycobacterium tuberculosis
isolated from patients with pulmonary tuberculosis in the Archangel Oblast, Russia, in 1998 and 1999 were studied by using restriction fragment length polymorphism (RFLP) analysis with the IS
6110
probe and spoligotyping. Resistance of the strains to antituberculosis drugs was analyzed by the BACTEC method, and mutations associated with rifampin resistance were detected by using the Inno-LiPA Rif. TB test. RFLP analysis and spoligotyping demonstrated that 53 (44.5%) of the strains belonged to the Beijing genotype. These strains showed a significantly higher rate of resistance than
M. tuberculosis
strains of other genotypes circulating in the region. In particular, 43.4% of the strains of the Beijing genotype were multidrug resistant; in contrast, only 10.6% of the other strains were. Of the strains of the Beijing genotype, 92.5% were part of a cluster, while only 33.3% of the remaining strains were clustered. Analysis of the medical records of the patients demonstrated that individuals infected with a strain of the Beijing genotype were significantly more likely to be alcohol abusers and to have chronic obstructive pulmonary disease prior to the tuberculosis diagnosis. Multivariate analysis showed that both variables were independently associated with infection by strains belonging to the Beijing genotype. Our study demonstrated that strains of the Beijing genotype are an important cause of tuberculosis in the Archangel Oblast and that dissemination of these strains is associated with the high incidence of drug resistance.
Automated Ribotyping of Vancomycin-Resistant
Enterococcus faecium
Isolates
Authors: Brisse Sylvain , Fussing Vivian , Ridwan Ben , Verhoef Jan , Willems Rob J. L.
Published: July 2002

Vancomycin-resistant
Enterococcus faecium
(VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes
Eco
RI and
Bam
HI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (
Eco
RI) and 16 (
Bam
HI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28
Eco
RI ribotypes, 25
Bam
HI ribotypes, and 28 sequence types. Ribotyping with
Eco
RI and
Bam
HI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the
esp
gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend
Eco
RI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.
H-1 Parvovirus-Associated Replication Bodies: a Distinct Virus-Induced Nuclear Structure
Authors: Cziepluch Celina , Lampel Stefan , Grewenig Annabel , Grund Christine , Lichter Peter , Rommelaere Jean
Published: July 2002

We have identified a nuclear structure that is induced after infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition, two cellular proteins were shown to accumulate in H1 PAR-bodies: (i) the proliferating cell nuclear antigen (PCNA) which is essential for chromosomal and parvoviral replication and (ii) the NS1-interacting small glutamine-rich TPR-containing protein (SGT), suggesting a role for the latter in parvoviral replication and/or gene expression. Since many DNA viruses target preexisting nuclear structures, known as PML-bodies, for viral replication and gene expression, we have determined the localization of H-1 PAR- and PML-bodies by double-fluorescence labeling and confocal microscopy and found them to be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize with other prominent nuclear structures such as nucleoli, coiled bodies, and speckled domains. Electron microscopy analysis revealed that NS1, as detected by indirect immunogold labeling, was localized in ring-shaped electron-dense nuclear structures corresponding in size and frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear bodies for DNA replication but rather induces the formation of a novel structure in the nucleus of infected cells.
Liver-Specific Alpha 2 Interferon Gene Expression Results in Protection from Induced Hepatitis
Authors: Aurisicchio Luigi , Delmastro Paola , Salucci Valentina , Paz Odalys Gonzalez , Rovere Patrizia , Ciliberto Gennaro , La Monica Nicola , Palombo Fabio
Published: July 2002

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-α). However, systemic delivery of r-hIFN-α is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-α antiviral efficacy, we have explored the therapeutic potential of murine IFN-α2 (mIFNα2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-α2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-α2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-α2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-α2.
Expression of Human Herpesvirus 6B
rep
within Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo
Authors: Mori Yasuko , Dhepakson Panadda , Shimamoto Takuya , Ueda Keiji , Gomi Yasuyuki , Tani Hideki , Matsuura Yoshiharu , Yamanishi Koichi
Published: July 2002

We have characterized the human herpesvirus 6B (HHV-6B)
rep
gene, which is a homologue of the adeno-associated virus type 2
rep
and is unique in the herpesvirus family. Three transcripts, 9.0, 5.0, and 2.7 kb (the major transcript), were detected by Northern blotting using an HHV-6B
rep
probe under late conditions. We investigated the expression kinetics of the
rep
gene using cycloheximide (CHX) and phosphonoformic acid (PFA), which are inhibitors of protein synthesis and viral DNA synthesis, respectively. The 5.2-kb transcript was mainly detected in the absence of protein biosynthesis upon infection, and none of the 9.0-, 5.0-, and 2.7-kb transcripts detected under the late conditions were detected in the presence of CHX and PFA. Sequences obtained from a cDNA library showed that the 5.0- and 2.7-kb transcripts were spliced from two and three exons, respectively, and the 2.7-kb transcript was more abundant. Immunohistochemistry using an antibody raised against the HHV-6
rep
gene product (REP) revealed that REP was mainly present in the nucleus of MT-4 cells within 24 h after infection with HHV-6B. Using pull-down assays, coimmunoprecipitation, and a mammalian two hybrid system, we showed that HHV-6 REP binds to a transcription factor, human TATA-binding protein, through its N-terminal region.
Cowpea Mosaic Virus Infection Induces a Massive Proliferation of Endoplasmic Reticulum but Not Golgi Membranes and Is Dependent on De Novo Membrane Synthesis
Authors: Carette Jan E. , Stuiver Marchel , Van Lent Jan , Wellink Joan , Van Kammen Ab
Published: July 2002

Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis.
Characteristics of the Adeno-Associated Virus Preintegration Site in Human Chromosome 19: Open Chromatin Conformation and Transcription-Competent Environment
Authors: Lamartina Stefania , Sporeno Elisabetta , Fattori Elena , Toniatti Carlo
Published: July 2002

Adeno-associated virus (AAV) establishes latency in infected cells by integrating into the cellular genome, with a high preference for a unique region, called AAVS1, of the human chromosome 19. The AAV proteins Rep78 and -68 are postulated to initiate the site-specific integration process by binding to a Rep binding site (RBS) in AAVS1. We provide further evidence to corroborate this model by demonstrating that the AAVS1 RBS in human cell lines is located near a DNase I hypersensitive “open” chromatin region and therefore is potentially easily accessible to Rep proteins. This open conformation is maintained in transgenic rats which carry an AAVS1 3.5-kb DNA fragment and are proficient for Rep-mediated site-specific integration. Interestingly, the core of the DNAse I hypersensitive site in AAVS1 corresponds to a sequence displaying transcriptional enhancer-like properties, suggesting that AAVS1 constitutes a transcription-competent environment. The implications of our findings for AAV physiology and gene therapy are discussed.
Regulation of the Epstein-Barr Virus C Promoter by AUF1 and the Cyclic AMP/Protein Kinase A Signaling Pathway
Authors: Fuentes-Pananá Ezequiel M. , Peng RongSheng , Brewer Gary , Tan Jie , Ling Paul D.
Published: July 2002

EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the expression of viral and cellular genes required for EBV-driven B-cell immortalization. Elucidating the mechanisms by which EBNA2 regulates viral and cellular gene expression is necessary to understand EBV-induced B-cell immortalization and viral latency in humans. EBNA2 targets to the latency C promoter (Cp) through an interaction with the cellular DNA binding protein CBF1 (RBPJk). The EBNA2 enhancer in Cp also binds another cellular factor, C promoter binding factor 2 (CBF2), whose protein product(s) has not yet been identified. Within the EBNA2 enhancer in Cp, we have previously identified the DNA sequence required for CBF2 binding and also determined that this element is required for efficient activation of Cp by EBNA2. In this study, the CBF2 activity was biochemically purified and microsequenced. The peptides sequenced were identical to the hnRNP protein AUF1. Antibodies against AUF1 but not antibodies to related hnRNP proteins reacted with CBF2 in gel mobility shift assays. In addition, stimulation of the cellular cyclic AMP (cAMP)/protein kinase A (PKA) signal transduction pathway results in an increase in detectable CBF2/AUF1 binding activity extracted from stimulated cells. Furthermore, the CBF2 binding site was able to confer EBNA2 responsiveness to a heterologous promoter when transfected cells were treated with compounds that activate PKA or by cotransfection of plasmids expressing a constitutively active catalytic subunit of PKA. EBNA2-mediated stimulation of the latency Cp is also increased in similar cotransfection assays. These results further support an important role for CBF2 in mediating EBNA2 transactivation; they identify the hnRNP protein AUF1 as a major component of CBF2 and are also the first evidence of acis-acting sequence other than a CBF1 binding element that is able to confer responsiveness to EBNA2.
Analysis of Mason-Pfizer Monkey Virus Gag Domains Required for Capsid Assembly in Bacteria: Role of the N-Terminal Proline Residue of CA in Directing Particle Shape
Authors: Rumlova-Klikova Michaela , Hunter Eric , Nermut Milan V. , Pichova Iva , Ruml Tomas
Published: July 2002

Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.
A Dual Infection/Competition Assay Shows a Correlation between Ex Vivo Human Immunodeficiency Virus Type 1 Fitness and Disease Progression
Authors: Quiñones-Mateu Miguel E. , Ball Sarah C. , Marozsan Andre J. , Torre Vincent S. , Albright Jamie L. , Vanham Guido , van der Groen Guido , Colebunders Robert L. , Arts Eric J.
Published: July 2002

This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4+T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r= 0.84,P= 0.033) and LTS (r= 0.86,P= 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.
The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA
Authors: Hayashi Yasuyuki , Kitamura Yoshiyuki , Nakanishi Mayumi , Koike Katsuro
Published: July 2002

In the replication cycle of hepadnavirus DNA, the double-stranded linear form of viral DNA is generated as a minor replicative intermediate, which is efficiently converted to covalently closed circular DNA (cccDNA) by intramolecular recombination (W. Yang and J. Summers, J. Virol. 69:4029–4036, 1995). We previously found a binding site of transcription factor Yin and Yang 1 (YY1) in one terminal region of the double-stranded linear replicative hepatitis B virus (HBV) DNA (M. Nakanishi-Matsui, Y. Hayashi, Y. Kitamura, and K. Koike, J. Virol. 74:5562–5568, 2000). However, it is not known whether the YY1-binding site is required for the intramolecular recombination of HBV DNA. In this study, we established an HBV-producing system in which the cccDNA appeared to be generated from the transfected linear DNA or the linear replicative DNA by nonhomologous end joining (NHEJ) or by both NHEJ and homologous recombination between terminally repeated sequences, respectively. When the YY1-binding site in the terminal region of transfected linear viral DNA was mutated, the cccDNA was generated merely by NHEJ. Results suggest that the YY1-binding site in the terminal region of linear replicative HBV DNA is required for intramolecular recombination between terminally repeated sequences.
Replication but Not Transcription of Simian Virus 40 DNA Is Dependent on Nuclear Domain 10
Authors: Tang Qiyi , Bell Peter , Tegtmeyer Peter , Maul Gerd G.
Published: July 2002

DNA viruses from several families including herpes simplex virus type 1, adenovirus type 5, and simian virus 40 (SV40), start their transcription and replication adjacent to a specific nuclear domain, ND10. We asked whether a specific viral DNA sequence determines the location of these synthetic activities at such restricted nuclear sites. Partial and overlapping SV40 sequences were introduced into a β-galactosidase expression vector, and the β-galactosidase transcripts were localized by in situ hybridization. Transcripts derived from control plasmids were found throughout the nucleus and at highly concentrated sites but not at ND10. SV40 genomic segments supported ND10-associated transcription only when the origin and the coding sequence for the large T antigen were present. When the large T-antigen coding sequence was eliminated but the T antigen was constitutively expressed in COS-7 cells, the viral origin was sufficient to localize transcription and replication to ND10. Deletion analysis showed that only the large T-antigen binding site II (the core origin) was required but the T antigen was needed for detectable transcription at ND10. Large T antigen expressed from plasmids without the viral core origin did not bind or localize to ND10. Blocking of DNA replication prevented the accumulation of transcripts at ND10, indicating that only sites with replicating templates accumulated transcripts. Transcription at ND10 did not enhance total protein synthesis of plasmid transcripts. These findings suggest that viral transcription at ND10 may only be a consequence of viral genomes directed to ND10 for replication. Although plasmid transcription can take place anywhere in the nucleus, T-antigen-directed replication is apparently restricted to ND10.
Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model
Authors: Yount Boyd , Curtis Kristopher M. , Baric Ralph S.
Published: July 2002

A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The ∼30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of ∼28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.
Testing the Hypothesis of a Recombinant Origin of Human Immunodeficiency Virus Type 1 Subtype E
Authors: Anderson Jon P. , Rodrigo Allen G. , Learn Gerald H. , Madan Anup , Delahunty Claire , Coon Michael , Girard Marc , Osmanov Saladin , Hood Leroy , Mullins James I.
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.
The Region between Amino Acids 245 and 265 of the Bovine Leukemia Virus (BLV) Tax Protein Restricts Transactivation Not Only via the BLV Enhancer but Also via Other Retrovirus Enhancers
Authors: Tajima Shigeru , Aida Yoko
Published: July 2002

Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type 1 (HTLV-1). The Tax protein of BLV acts through the 5′ long terminal repeat (LTR) of BLV and activates the transcription of BLV. In this study, we amplified
tax
genes from BLV-infected cattle using PCR. We cloned the genes and monitored the transcriptional activities of the products. Seven independent mutant Tax proteins, with at least one amino acid substitution between residues 240 and 265, exhibited a markedly stronger ability to stimulate the viral LTR-directed transcription than the wild-type Tax protein. Analysis of chimeric Tax proteins derived from wild-type and mutant Tax proteins clearly demonstrated that a single substitution between residue 240 and 265 might be critical for the higher activities of the Tax mutant proteins. Furthermore, it appeared that transient expression of a Tax mutant protein was better able to increase the production of viral proteins and particles from a defective recombinant proviral clone of BLV than was wild-type Tax. Analysis of mutations within the U3 region of the LTR revealed that a cyclic AMP-responsive element in Tax-responsive element 2 might be sufficient for the enhanced activation mediated by the mutant proteins. In addition to the LTR of BLV, other viral enhancers, such as the enhancers of HTLV-1 and of mouse mammary tumor virus, which cannot be activated by wild-type BLV Tax protein, were activated by a Tax mutant protein. Our observations suggest that the transactivation activity and target sequence specificity of BLV Tax might be limited or negatively regulated by the region of the protein between amino acids 240 and 265.
Structural and Functional Analysis of the
Xestia c-nigrum
Granulovirus Matrix Metalloproteinase
Authors: Ko Rinkei , Okano Kazuhiro , Maeda Susumu
Published: July 2002

Sequence analysis of the
Xestia c-nigrum
granulovirus (XcGV) genome identified an open reading frame encoding a 469-amino-acid (54-kDa) protein with over 30% amino acid sequence identity to a region of about 150 amino acids that includes the catalytic domains of human stromelysin 1 (Str1)/matrix metalloproteinase 3 (MMP-3) (EC
3.4.24.17
) and sea urchin hatching enzyme (HE). Stromelysin homologs have not been reported from baculoviruses or other viruses. Unlike human Str1 and sea urchin HE, the putative XcGV-MMP does not have a signal peptide and lacks the peptide motif involved in the cysteine switch that maintains other MMPs in an inactive form. The putative XcGV-MMP, however, possesses a conserved zinc-binding motif in its putative catalytic domain. The XcGV-MMP homolog was cloned, and a recombinant
Bombyx mori
nucleopolyhedrovirus (BmNPV) that expresses XcGV-MMP under the polyhedrin promoter was constructed. A distinct pattern of melanization was observed in
B. mori
larvae infected with MMP-expressing BmNPV. Fat body extracts from larvae overexpressing the 54-kDa recombinant MMP digested dye-impregnated collagen (Azocoll). The enzymatic activity was inhibited by two metalloproteinase inhibitors, EDTA and 1,10-phenanthroline. These results suggest that the XcGV MMP-3 gene homolog encodes a functional metalloproteinase.
Interaction between Herpes Simplex Virus Type 1 IE63 Protein and Cellular Protein p32
Authors: Bryant Helen E. , Matthews David A. , Wadd Sarah , Scott James E. , Kean Joy , Graham Susan , Russell William C. , Clements J. Barklie
Published: July 2002

The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes. One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts. We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H. Bryant et al., J. Biol. Chem. 274:28991–28998, 1999). Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32. Confirmation of this interaction was provided by coimmunoprecipitation from virus-infected cells and recombinant p32 binding assays. A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2. Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells. As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing. As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts.
Interaction between Hepatitis B Virus Core Protein and Reverse Transcriptase
Authors: Lott Lisa , Beames Burton , Notvall Lena , Lanford Robert E.
Published: July 2002

Previous mutagenesis studies with hepatitis B virus (HBV) suggest that continued interactions with core are required for several steps in genomic replication. To examine core-polymerase (Pol) interactions, insect cells were coinfected with baculovirus constructs that independently expressed core and Pol. The results demonstrated several features with implications that core plays an interactive role with HBV Pol: (i) core coprecipitated with constructs expressing full-length Pol as well as the terminal protein (TP), reverse transcriptase (RT) and RNase H domains of Pol, independently; (ii) coprecipitation of core was not dependent on the presence of an epsilon stem-loop sequence; and (iii) core-Pol complexes migrated as intact capsid particles, as detected by sucrose gradient analysis. To analyze the structural and sequence requirements of core in recognition of Pol, a series of core mutants with two- to four-amino-acid insertions or carboxy-terminal deletions were assessed for Pol interaction. The results indicated that capsid formation is required but not sufficient for interaction with Pol and that the TP and RT domains of Pol have different requirements for interaction with core. To map the core binding sites on Pol, a panel of amino- and carboxy-terminal deletion mutants of the TP and RT domains of Pol were analyzed for interaction with core. At least three separate core binding sites on Pol were detected. This analysis begins to define basic requirements for core-Pol interactions, but further study is necessary to delineate the effects of these interactions on encapsidation and genome replication.
Genetic Manipulation of Arterivirus Alternative mRNA Leader-Body Junction Sites Reveals Tight Regulation of Structural Protein Expression
Authors: Pasternak Alexander O. , Gultyaev Alexander P. , Spaan Willy J. M. , Snijder Eric J.
Published: July 2002

To express its structural proteins, the arterivirus
Equine arteritis virus
(EAV) produces a nested set of six subgenomic (sg) RNA species. These RNA molecules are generated by a mechanism of discontinuous transcription, during which a common leader sequence, representing the 5′ end of the genomic RNA, is attached to the bodies of the sg RNAs. The connection between the leader and body parts of an mRNA is formed by a short, conserved sequence element termed the transcription-regulating sequence (TRS), which is present at the 3′ end of the leader as well as upstream of each of the structural protein genes. With the exception of RNA3, only one body TRS was previously assumed to be used to join the leader and body of each EAV sg RNA. Here we show that for the synthesis of two other sg RNAs, RNA4 and RNA5, alternative leader-body junction sites that differ substantially in transcriptional activity are used. By site-directed mutagenesis of an EAV infectious cDNA clone, the alternative TRSs used to generate RNA3, -4, and -5 were inactivated, which strongly influenced the corresponding RNA levels and the production of infectious progeny virus. The relative amounts of RNA produced from alternative TRSs differed significantly and corresponded to the relative infectivities of the virus mutants. This strongly suggested that the structural proteins that are expressed from these RNAs are limiting factors during the viral life cycle and that the discontinuous step in sg RNA synthesis is crucial for the regulation of their expression. On the basis of a theoretical analysis of the predicted RNA structure of the 3′ end of the EAV genome, we propose that the local secondary RNA structure of the body TRS regions is an important factor in the regulation of the discontinuous step in EAV sg mRNA synthesis.
Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro
Authors: Osman T. A. M. , Hemenway C. L. , Buck K. W.
Published: July 2002

A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.
Functional Role of pX Open Reading Frame II of Human T-Lymphotropic Virus Type 1 in Maintenance of Viral Loads In Vivo
Authors: Bartoe Joshua T. , Albrecht Björn , Collins Nathaniel D. , Robek Michael D. , Ratner Lee , Green Patrick L. , Lairmore Michael D.
Published: July 2002

Human T-lymphotropic virus type 1
(HTLV-1) causes adult T-cell leukemia/lymphoma and is associated with a variety of immune-mediated disorders. The role of four open reading frames (ORFs), located between
env
and the 3′ long terminal repeat of HTLV-1, in mediating disease is not entirely clear. By differential splicing, ORF II encodes two proteins, p13
II
and p30
II
, both of which have not been functionally defined. p13
II
localizes to mitochondria and may alter the configuration of the tubular network of this cellular organelle. p30
II
localizes to the nucleolus and shares homology with the transcription factors Oct-1 and -2, Pit-1, and POU-M1. Both p13
II
and p30
II
are dispensable for infection and immortalization of primary human and rabbit lymphocytes in vitro. To test the role of ORF II gene products in vivo, we inoculated rabbits with lethally irradiated cell lines expressing the wild-type molecular clone of HTLV-1 (ACH.1) or a clone containing selected mutations in ORF II (ACH.30/13.1). ACH.1-inoculated animals maintained higher HTLV-1-specific antibody titers than animals inoculated with ACH.30/13.1. Viral p19 antigen was transiently detected in ex vivo cultures of peripheral blood mononuclear cells (PBMC) from only two ACH.30/13.1-inoculated rabbits, while PBMC cultures from all ACH.1-inoculated rabbits routinely produced p19 antigen. In only three of six animals exposed to the ACH.p30
II
/p13
II
clone could provirus be consistently PCR amplified from extracted PBMC DNA and quantitative competitive PCR showed the proviral loads in PBMC from ACH.p30
II
/p13
II
-infected rabbits to be dramatically lower than the proviral loads in rabbits exposed to ACH. Our data indicate selected mutations in pX ORF II diminish the ability of HTLV-1 to maintain high viral loads in vivo and suggest an important function for p13
II
and p30
II
in viral pathogenesis.
Biochemical and Structural Analysis of Isolated Mature Cores of Human Immunodeficiency Virus Type 1
Authors: Welker Reinhold , Hohenberg Heinrich , Tessmer Uwe , Huckhagel Carola , Kräusslich Hans-Georg
Published: July 2002

Mature human immunodeficiency virus type 1 (HIV-1) particles contain a cone-shaped core structure consisting of the internal ribonucleoprotein complex encased in a proteinaceous shell derived from the viral capsid protein. Because of their very low stability after membrane removal, HIV-1 cores have not been purified in quantities sufficient for structural and biochemical analysis. Based on our in vitro assembly experiments, we have developed a novel method for isolation of intact mature HIV-1 cores. Concentrated virus suspensions were briefly treated with nonionic detergent and immediately centrifuged in a microcentrifuge for short periods of time. The resuspended pellet was subsequently analyzed by negative-stain and thin-section electron microscopy and by immunoelectron microscopy. Abundant cone-shaped cores as well as tubular and aberrant structures were observed. Stereo images showed that core structures preserved their three-dimensional architecture and exhibited a regular substructure. Detailed analysis of 155 cores revealed an average length of ca. 103 nm, an average diameter at the base of ca. 52 nm, and an average angle of 21.3°. There was significant variability in all parameters, indicating that HIV cores are not homogeneous. Immunoblot analysis of core preparations allowed semiquantitative estimation of the relative amounts of viral and cellular proteins inside the HIV-1 core, yielding a model for the topology of various proteins inside the virion.