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PubMed2001

Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients.

Posttransplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplantation recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per mL blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3.4% of the whole blood samples was above the cut-off value (P <.0001) despite heavy immune suppression and cytomegalovirus (CMV)-related disease. These findings illustrate the clinical importance of frequent EBV DNA load monitoring in LTx recipients. The increased EBV DNA loads in PTLD patients were restricted to the cellular blood compartment, as parallel serum samples were all below cut-off value, which indicates absence of lytic viral replication. EBV(+) cells in PTLD patients have a very short doubling time, which can be as low as 56 hours, thereby creating the need for high screening frequency in high-risk patients. Furthermore, it is shown that EBV and CMV can reactivate independently in LTx recipients and that EBV DNA load monitoring may be useful in discriminating PTLD from rejection.

Adult↗
PubMed2026

Bacteria and phage consortia modulate cecal SCFA production and host metabolism to enhance feed efficiency in ducks.

BACKGROUND: The gut microbiota influences poultry health, nutrition, feed efficiency (FE), and overall productivity. However, the relationship between gut microbes, including bacteria and phages, and FE in ducks remains underexplored. To address this, we integrated cecal 16S amplicon, metagenome, microbiota-derived short-chain fatty acids (SCFAs) profiling, liver transcriptome, and serum metabolome data to illustrate the contribution of the gut microbiome (bacteria and viruses) to duck FE. RESULTS: We reconstructed viral genomes and prokaryotic metagenome-assembled genomes (MAGs) and annotated their genes using comprehensive databases. Prokaryotic hosts of viruses were also predicted to understand virus-host dynamics within the gut ecosystem. Our results revealed that high-FE ducks have higher concentration of propionate and butyrate in cecum compared with low-FE ducks. The metagenome sequencing revealed distinct cecal microbiota profiles between two groups, with increased relative abundance of representative SCFA producers, especially Paraprevotella sp905215575 and Bacteroides sp944322345, and enhanced SCFA-biosynthesis pathways in high-FE ducks. Virome genome assembly identified two phages encoding auxiliary metabolic genes (AMGs) involved in pyruvate metabolism, enhancing nutrient availability for host bacteria to produce SCFAs (e.g., temperate phage-encoded pyruvate phosphate dikinase) or exploiting host central metabolic pathways for viral replication (e.g., lytic phage-encoded formate C-acetyltransferase). Furthermore, these representative SCFA-producing bacteria and phage consortia were associated with serum metabolites (including L-histidine and 4-hydroxydecanedioylcarnitine) linked to duck FE. CONCLUSION: Collectively, these findings provide novel insights into the gut microbial factors regulating FE in ducks, offering potential strategies to optimize poultry nutrition and productivity. Video Abstract.

Animals↗
PubMed2006

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed.

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.

Cell Line↗
PubMed2026

Genomic and functional characterization of novel therapeutic lytic bacteriophages targeting multidrug-resistant Enterobacter cloacae.

The alarming rates at which extensively drug-resistant (XDR) and pandrug-resistant (PDR) Enterobacter cloacae in hospitals are increasing has begun to severely limit treatment options, and thus the urgency for alternative interventions, including bacteriophage therapy. The purpose of the study was to isolate and molecularly characterize phages that can infect E. cloacae, and, furthermore, to assess the antimicrobial efficacy of the four novel lytic bacteriophages (MMRP1, MMRP2, MMRP3, and MMRP4) against antimicrobial-resistant E. cloacae isolates and to evaluate their potential as alternative therapeutic strategies. These novel phages were characterized by plaque morphology, transmission electron microscopy (TEM), host range testing, thermal and chloroform stability assays, bacterial reduction assays, and whole-genome sequencing (WGS). Among 27 clinical isolates, MDR, XDR, and PDR phenotypes were observed in 20 (74.1%), six (22.2%), and one (3.7%) isolates, respectively. All four phages produced clear lytic plaques (0.5-3.0&#x202f;mm) with titers reaching up to 6&#x202f;&#xd7;&#x202f;1010 PFU/mL, and the phage cocktail lysed 81.4% (22 of 27 isolates) of clinical isolates with high host specificity. TEM revealed that all four E. cloacae-infecting phages (MMRP1, MMRP2, MMRP3, and MMRP4) belong to the class Caudoviricetes, exhibiting icosahedral capsids, tailed morphology, and double-stranded DNA genomes, consistent with current ICTV classification criteria. Whole genome sequencing and comparative phylogenetic analysis further resolved the taxonomic placement of these phages at the family level, positioning MMRP1 within the family Demerecviridae and MMRP4 within the family Straboviridae. All phages were stable from -20 to 40&#x202f;&#xb0;C and were unaffected by exposure to chloroform. Phage cocktail reduced bacterial OD&#x2086;&#x2080;&#x2080; to &#x2264; 0.3 within 4&#x202f;h in the bacterial reduction test. WGS revealed large circular dsDNA genomes of ~132 kbp (MMRP1) and ~149 kbp (MMRP4), GC content of 38%, and modular architectures encoding structural, lytic, and replication gene modules. The most striking and highlighted suggestion that in vitro evaluation of MMRP1 and MMRP4 are highly recommended to more deeper future experimental studies to combat MDR E. cloacae nosocomial infections supported by genomic foundation and eventually, the possibility to be suitable for phage-engineering applications in clinical settings.

Enterobacter cloacae↗
PubMed2001

Serum/plasma viral DNA: mechanisms and diagnostic applications to nasopharyngeal and cervical carcinoma.

Following reports describing circulating tumor DNA, serum/plasma viral nucleic acid has shown its potential as a new diagnostic target in cancer. In the majority of examples of viral carcinogenesis, the viral genome is consistently present in certain tumors and serves as an effective marker. This article reviews recent findings, proposes possible mechanisms, and examines the potential clinical application of serum/plasma Epstein-Barr virus (EBV) DNA in nasopharyngeal cancer (NPC) and human papillomavirus (HPV) DNA in cervical carcinoma (CC). These tumors share a DNA viral etiology and present similar histopathological findings. However, plasma EBV and HPV DNA are distinct in several aspects, including incidence, mechanism of release from tumor, and clinical application. Both circulating cell-free EBV and HPV DNA reveal the same viral type as their matched tumors, indicating both are derived from the neoplastic tissue. Plasma viral DNA incidence and copy number are high in NPC, but low in HPV-associated cancers. Whereas much EBV DNA in NPC is episomal, the resistance to DNase treatment of serum EBV DNA and evidence confirming lytic EBV replication in NPC suggest that a reasonable proportion of plasma EBV DNA is virions. On the contrary, plasma HPV genomes, as in CC, integrate into host chromosome. Plasma EBV DNA copy number, by quantitative PCR, is related to tumor mass, predicts prognosis, measures immediate response to treatment, and is useful in early detection of recurrence. Plasma HPV DNA, on the other hand, is associated with and can be considered as an early tumor marker for distant metastasis.

Biomarkers, Tumor↗
PubMed2003

Primary nasopharyngeal non-Hodgkin lymphoma and its relationship with Epstein-Barr virus infection.

OBJECTIVES: To investigate the immunophenotypes of primary nasopharyngeal non-Hodgkin lymphoma (NPL) and their relationship to Epstein-Barr virus (EBV) infection. METHODS: The clinical data and biopsies of 73 patients with NPL were collected in Guangzhou. In situ hybridization was performed to detect the EBV-encoded small non-polyadenylated nuclear RNAs (EBERs) on biopsy slides. Immunohistochemistry was used to classify the immunophenotypes of NPL and detect EBV antigen expression. RESULTS: Forty-four (60.27%) of the 73 NPLs were of B cell lineage (CD79alpha(+)/CD3(-)/CD56(-)) while the 29 others (39.73%) were of non-B cell lineage. Seventy-three NPLs could be classified into 3 major immunophenotypes: B cell (CD79alpha(+)/CD3(-)/CD56(-), 44 cases), peripheral T cell (CD79alpha(-)/CD3(+)/CD56(-), 22) and NK/T cell (CD79alpha(-)/CD3(+)/CD56(+), 7). The percentages of EBV infection differed among the 3 major immunophenotypes (B cell: 11.36%, 5/44; peripheral T cell: 81.82%, 18/22; NK/T cell: 100%, 7/7). Both CD56(-) positive and CD56(-) negative immunophenotypes could further be divided into 4 subtypes: CD8(-)/CD4(-), CD8(+)/CD4(-), CD8(-)/CD4(+) and CD8(+)/CD4(+). All the CD8(-)/CD4(-) NPLs with CD56(-) positivity (7) or CD56(-) negativity (2) were infected with EBV. The neoplastic cells of a nasopharyngeal Burkitt's lymphoma expressed EBV nuclear antigen 1 (EBNA1) and EBV RNA (EBERs) only. In the other 29 EBV-infected NPLs, most of the lymphoma cells harboring EBV also expressed EBNA1 and EBERs; 21 of the 29 NPLs had a considerable number of neoplastic cells expressing latent membrane protein 1 (LMP1) (21/29, 72.41%) and 23 of 29 NPLs expressed latent membrane protein 2A (LMP2A) (23/29, 79.31%). A few lymphoma cells in 17 (17/29, 58.62%), 23 (23/29, 79.31%) and 22 NPLs (22/29, 75.86%) expressed Zta (Bam HI Z transactivator), viral capsid antigen (VCA) and membrane antigen (MA), respectively. CONCLUSIONS: The prevalence ratio of the 3 immunophenotypes, namely, B cell, peripheral T cell and NK/T cell lymphoma, is about 6:3:1. However, the EBV infection ratio is reversed, 1:8:10. All the NK/T cell (CD56(+)) and peripheral immature T cell (CD3(+)/CD8(-)/CD4(-)) NPLs were EBV-infected. Except for one Burkitt's lymphoma, the EBV harbored in both B cell and non-B cell NPLs was mainly latent infection, type II, expressing EBNA1, LMP1 and LMP2A. However, the EBV found in a few lymphoma cells could become replicative, expressing lytic proteins.

Adolescent↗
PubMed1989

Mouse hepatitis virus 3 pathogenicity expressed by a lytic viral infection in bone marrow 14.8+ mu+ B lymphocyte subpopulations.

Mouse hepatitis virus type 3 (MHV3) provides an excellent model for studying viral-B lymphocyte interaction in the immune system, which plays an important role in the outcome of an acute disease. Bone marrow B lymphocyte subpopulations, at various times postinfection, were studied in genetically C57BL/6 and resistant A/J mice, infected with pathogenic L2-MHV3 and its nonpathogenic variant, YAC-MHV3. B lineage cell subpopulations were identified by double immunofluorescence assays using mAb of terminal deoxynucleotidyl transferase, 14.8 and cytoplasmic (cu) or surface (su) Ig mu-chains. Results revealed diminished percentage and absolute number in the bone marrow 14.8+ mu+ B lymphocyte subpopulations, including pre-B (cu+ su-) and B (cu+ su+) cells of L2-MHV3-infected susceptible C57BL/6 mice; whereas, slight or no increase was evident in the cell subpopulations of L2-MHV3 infected resistant A/J mice or in YAC-MHV3 infected in both strains of mice. Abnormal large-sized forms of the 14.8+ mu+ cells occurred, at 48-h postinfection, in L2-MHV3-infected susceptible C57BL/6 mice only. In contrast, no change in the percentage and absolute number of precursor cells (terminal deoxynucleotidyl transferase positive) and pre pre-B cells (14.8+ mu-) were detected in all infected mice. In vitro L2-MHV3 infection of C57BL/6 bone marrow purified B lineage cell subpopulations showed that pre-B (cu+ su-) and B (cu+ su+) cells became abnormally large in size and depleted in number as a result of a productive and lytic viral replication. Low L2-MHV3 viral replication occurred in these cell subpopulations of A/J mice but no YAC-MHV3 virus was produced in the cells of both strains of mice. Pre pre-B (14.8+ mu-) cells in both strains were not permissive to L2-MHV3 or YAC-MHV3 viral replication. These results are discussed with regard to the role of humoral immunodeficiency in the pathogenic process.

Acute Disease↗
PubMed1993

Study of the mechanisms by which CD4+ T cells contribute to protection in Theiler's murine encephalomyelitis.

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus which causes a biphasic central nervous system (CNS) disease in certain strains of mice. Lytic virus replication within the CNS causes acute damage at early times post-infection, with the surviving animals developing a chronic CNS demyelinating disease. This damage is thought to result both from direct viral damage and from an immunopathological CD4+ T-cell mediated delayed-type hypersensitivity response to virus. By contrast, CD4+ T cells have a vital protective role at early times post-infection, as mice specifically depleted of CD4+ T cells of this subset prior to infection with TMEV die within 3-5 weeks. In an investigation of how CD4+ T cells act to mediate protection in TMEV-infected mice, we show that CD4+ cell-depleted animals, which fail to make a significant antiviral antibody response, could be protected by passive transfer of neutralizing antibodies. However, surviving animals had high levels of persisting virus in the CNS and they developed very severe symptoms of chronic demyelinating disease. The appearance of infectious virus was not due to selection of neutralizing antibody-resistant viral variants. These results demonstrate that the key protective role of CD4+ T cells in TMEV-infected mice is to provide help for antibody production by B cells at early times post-infection, but that other CD4+ cell-dependent mechanisms must contribute to control of virus replication, and are of importance in determining the levels of virus subsequently persisting in the CNS, and hence the severity of the chronic demyelinating disease.

Animals↗
PubMed1996

Virus-cell interactions in a natural killer-like cell line from a patient with lymphoblastic lymphoma.

Lymphoproliferative disorders involving Epstein-Barr virus (EBV) infected natural killer (NK) cells are reported with increasing frequency, but the nature and role of EBV infection in these cells remains undefined. In this study, we have investigated virus-cell interactions in the EBV-positive YTN10 cell line, an NK-like cell line established from a patient with lymphoblastic lymphoma. Low level expression of the EBV receptor CD21 molecule was detected by FACS and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Immunoblotting and RT-PCR analysis identified a latency II pattern of EBV gene expression, consisting of EBNA-1 transcription from the Qp promoter, in the absence of other EBNA gene expression, and accompanied by LMP-1 and LMP-2A expression. The EBV genome was present in episomal form and there was evidence for lytic viral replication. This latency pattern is typical of EBV gene expression in nasopharyngeal carcinoma and Hodgkin's disease, and differs from the full spectrum of EBV latent gene expression in most posttransplant lymphoproliferative disorders and from the restricted EBNA-1 expression in Burkitt's lymphoma tissues. The interaction between EBV and NK cells described here has important implications for the pathogenesis and treatment of EBV-infected NK malignancies.

Adolescent↗
PubMed1995

The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon.

Bacteriophage P1↗
PubMed1997

A particular DNA structure is required for the function of a cis-acting component of the Epstein-Barr virus OriLyt origin of replication.

OriLyt, thecis-acting element of Epstein-Barr virus lytic origin of replication, consists of upstream and downstream components. The upstream component plays a dual role in transcription and replication. The downstream component contains a homopurine-homopyrimidine sequence which forms an H palindrome. We show that the downstream component can adopt a triple helix structure in vitro, that the 5' border of the homopyrimidine sequence is sensitive to P1 nuclease when carried by a supercoiled plasmid and that an oligonucleotide complementary to the homopyrimidine strand is taken up by a plasmid carrying the OriLyt H palindrome. We also show that all mutations which alter the H palindrome impair both oligonucleotide uptake and OriLyt-dependent replication. Interestingly, compensatory mutations which restore an H palindrome also restore oligonucleotide uptake by the mutated plasmids and their OriLyt-dependent replication. Thus, there is a strong correlation between the inability of the OriLyt H palindrome to form a non-B-DNA structure in vitro and impairment of OriLyt-dependent replication. This suggests that the presence of a non-B-DNA structure in the OriLyt downstream component is required for OriLyt-dependent replication.

Base Sequence↗
PubMed1992

trans-acting requirements for replication of Epstein-Barr virus ori-Lyt.

Epstein-Barr virus (EBV) utilizes a completely different mode of DNA replication during the lytic cycle than that employed during latency. The latency origin of replication, ori-P, which functions in the replication of the latent episomal form of the EBV genome, requires only a single virally encoded protein, EBNA-1, for its activity. During the lytic cycle, a separate origin, ori-Lyt, is utilized. Relatively little is known about the trans-acting proteins involved in ori-Lyt replication. We established a cotransfection-replication assay to identify EBV genes whose products are required for replication of ori-Lyt. In this assay, a BamHI-H plasmid containing ori-Lyt was replicated in Vero cells cotransfected with the BamHI-H target, the three EBV lytic-cycle transactivators Zta, Rta, and Mta, and the EBV genome provided in the form of a set of six overlapping cosmid clones. By removing individual cosmids from the cotransfection mixture, we found that only three of the six cosmids were necessary for ori-Lyt replication. Subcloning of the essential cosmids led to the identification of six EBV genes that encode replication proteins. These genes and their functions (either known or predicted on the basis of sequence comparison with herpes simplex virus) are BALF5, the DNA polymerase; BALF2, the single-stranded DNA-binding protein homolog; BMRF1, the DNA polymerase processivity factor; BSLF1 and BBLF4, the primase and helicase homologs; and BBLF2/3, a potential homolog of the third component of the helicase-primase complex. In addition, ori-Lyt replication in this cotransfection assay was also dependent on one or more genes provided by the EBV SalI-F fragment and on the three lytic-cycle transactivators Zta, Rta, and Mta.

Amino Acid Sequence↗
PubMed2005

Activation of Kaposi's sarcoma-associated herpesvirus lytic gene expression during epithelial differentiation.

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1alpha promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.

Cell Differentiation↗
PubMed1994

Epstein-Barr virus-infected marmoset cells do not form lymphomas in mice with severe combined immunodeficiency.

EBV has been associated with several malignancies in humans. EBV can also infect marmoset B lymphocytes, which, as opposed to human B cells, are permissive for lytic Epstein-Barr viral replication. Mice with a severe combined immunodeficiency phenotype (SCID mice) are extremely susceptible to EBV-induced lymphomagenesis when inoculated with EBV-infected lymphocytes. We inoculated SCID mice with human and marmoset lymphoblastoid cells infected with the same EBV isolates. The marmoset cells never gave rise to lymphomas, even after the administration of acyclovir or an anti-natural killer cell antibody and observation periods of up to 16 wk. In contrast, the human lymphoblastoid cells nearly always gave rise to lymphomas within 8 wk. Furthermore, human lymphoblastoid cells genetically engineered to permit lytic EBV replication also readily formed tumors in the SCID mouse. Thus, in this system, it is the cellular milieu that is crucial in determining whether a given lymphoblastoid cell will give rise to a tumor, not the EBV isolate harbored by the cell or whether the virus is permitted to undergo lytic replication.

Animals↗
PubMed1998

Overexpression, purification and helix-destabilizing properties of Epstein-Barr virus ssDNA-binding protein.

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.

Animals↗
PubMed1987

Two major replicating simian virus 40 chromosome classes. Synchronous replication fork movement is associated with bound large T antigen during elongation.

We have analyzed the asynchronous progression of replication forks through the early (E) and late (L) gene sides in bidirectionally replicating SV40 chromosomes during lytic infection. By cutting purified replicating DNA with an appropriate single-site restriction endonuclease and measuring the contour lengths of replicated and unreplicated segments by electron microscopy, the positions of the two replication forks in each elongating intermediate were determined. Our results indicate that there are at least two major classes of replicating SV40 chromosomes which differ in their relative rate of E and L fork movement, the presence or absence of bound SV40 large T antigen during elongation, and the termination region utilized. These two classes also have altered apparent start sites for initiating bidirectional replication, flanking either side of core ori. The largest group (67%) replicated synchronously was associated with T antigen during elongation, appeared to initiate bidirectional elongation at nucleotide 5203 or 41 base pairs (bp) toward the E side of 0/5243, at the junction of T binding site I and ori, and terminated at the typical region centered at 0.5 map units. A second group (24%) replicated asynchronously with the L fork moving 3 times faster than the E fork, was not associated with T antigen during elongation, and terminated at a broad region centered at 0.73 map units. This group appeared to initiate at nucleotide 29 at the junction of the AT-rich region of ori, T binding site I, and the start of the 21-bp repeated transcriptional control sequences. A third group (9%) appeared to initiate at nucleotide 5148 or 95 bp to the E side of 0/5243 and replicated asynchronously preferentially on the E side at early times. However, this group is related to the synchronous class in that it contains bound T antigen and both forks move synchronously past 30% elongation, terminating at the same region. The association of T antigen with synchronous but not asynchronous DNA molecules indicates that T functions in regulating fork movement during elongation. A synchronization role implies that both forks are closely associated with one another in replicating molecules with bound T. Replicating molecules lacking T not only elongated highly asynchronously but preferential fork progression occurred almost exclusively on the L side. The ori region in asynchronous compared to synchronous intermediates was differentially sensitive to BglI digestion, indicating that nuclease digestion can distinguish between different populations of replicating molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Polyomavirus Transforming↗
PubMed2006

Amino acids in the basic domain of Epstein-Barr virus ZEBRA protein play distinct roles in DNA binding, activation of early lytic gene expression, and promotion of viral DNA replication.

The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding to DNA, in activating Rta, in stimulating early lytic gene expression, and in promoting viral DNA replication and viral late gene expression. These results are discussed in relationship to the recently solved crystal structure of ZEBRA bound to an AP-1 site.

Base Sequence↗
PubMed1996

Pathogenesis of ruminant herpesvirus infections.

Ruminants are hosts for members of both Alpha- and Gamma-herpesvirinae. A wide range of disease syndromes is associated with infections by these agents. The associated diseases reflect the biological nature of the causative viruses. Clinically, the symptoms may be mild and localized or include severe generalized disease, leading eventually to death. Much knowledge has been gained concerning the pathogenesis of some alpha-herpesviruses. Initially, these viruses replicate in epithelial cells at the portal of entry. The symptoms of the acute diseases are often associated with the destruction of those epithelial cells. However, as in the case of bovine herpesvirus 1 (BHV-1), the virus may spread in the infected host by viremia, gaining access to a broader range of tissues and organs, and causing a broader variety of diseases. Furthermore, many herpesviruses are capable of entering neuronal cells. There, they may replicate, which may lead to neuronal diseases, for example, encephalitis. In addition, the herpesviruses may establish latency in neuronal or lymphoid cells. During latency, apparently no viral antigens are synthesized but the genomes of the latent viruses are present in the nuclei of long living cells, such as, e.g., neurones of the ganglia corresponding to the sites of peripheral replication. Upon reactivation, the viruses re-establish the lytic cycle of replication. Shielded from the effectors of the immune system, they migrate back to the peripheral tissues where they are excreted and may be transmitted. Although a strong immune response is provoked during primary viral replication, these mechanisms help the herpesviruses to escape from immune surveillance during latency and to a lesser degree during reactivation. It has been observed that certain herpesviruses may behave differently upon infection of different hosts. Relatively little progress has been made concerning the understanding of the pathogenesis of ruminant herpesviruses but much has been learned about viral molecular biology. Many viral proteins have been identified and characterized and the technology to create recombinant viruses has been established. With these tools in our hands, it is now possible to address the really interesting questions concerning pathogenesis. We postulate that herpesviruses contain at least two sets of genes, a first set involved in gene expression and viral replication, and a second set responsible for functions, which may affect pathogenesis, latency, and virus/host interactions. Using recombinant virus technology, it will be possible in the future to design targeted deletions and gene transfers in ruminant herpesviruses in order to study the viral and host factors involved in pathogenesis on the molecular level.

Alphaherpesvirinae↗