scholarly journals Evaluation of the Lytic Origins of Replication of Kaposi's Sarcoma-Associated Virus/Human Herpesvirus 8 in the Context of the Viral Genome

2006 ◽  
Vol 80 (19) ◽  
pp. 9905-9909 ◽  
Author(s):  
Yiyang Xu ◽  
Alicia Rodriguez-Huete ◽  
Gregory S. Pari
Keyword(s):  
Viral Genome ◽  
Human Herpesvirus ◽  
Artificial Chromosome ◽  
Open Reading Frames ◽  
Human Herpesvirus 8 ◽  
Viral Dna ◽  
Herpesvirus 8 ◽  
Replication Assay ◽  
Transient Replication Assay ◽  
Reading Frames

ABSTRACT The lytic origins of DNA replication for human herpesvirus 8 (HHV8), oriLyt-L and oriLyt-R, are located between open reading frames K4.2 and K5 and ORF69 and vFLIP, respectively. These lytic origins were elucidated using a transient replication assay. Although this assay is a powerful tool for identifying many herpesvirus lytic origins, it is limited in its ability to evaluate the activity of replication origins in the context of the viral genome. To this end, we investigated the ability of a recombinant HHV8 bacterial artificial chromosome (BAC) to replicate in the absence of oriLyt-R, oriLyt-L, or both oriLyt regions. We generated the HHV8 BAC recombinants (BAC36-ΔOri-R, BAC36-ΔOri-L, and BAC36-ΔOri-RL), which removed one or all of the identified lytic origins. An evaluation of these recombinant BACs revealed that oriLyt-L was sufficient to propagate the viral genome, whereas oriLyt-R alone failed to direct the amplification of viral DNA.

scholarly journals Characterization of Human Herpesvirus 8 ORF59 Protein (PF-8) and Mapping of the Processivity and Viral DNA Polymerase-Interacting Domains

2000 ◽  
Vol 74 (23) ◽  
pp. 10920-10929 ◽  
Author(s):  
Szeman Ruby Chan ◽  
Bala Chandran
Keyword(s):  
Dna Polymerase ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Dependent Manner ◽  
Cell Nuclei ◽  
Viral Dna ◽  
Herpesvirus 8 ◽  
Barr Virus

ABSTRACT Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118–126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.

scholarly journals A Rhesus Macaque Rhadinovirus Related to Kaposi’s Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Encodes a Functional Homologue of Interleukin-6

1999 ◽  
Vol 73 (7) ◽  
pp. 6177-6181 ◽  
Author(s):  
Johnan A. R. Kaleeba ◽  
Eric P. Bergquam ◽  
Scott W. Wong
Keyword(s):  
Interleukin 6 ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frames ◽  
Human Herpesvirus 8 ◽  
Dependent Manner ◽  
Herpesvirus 8 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The rhesus rhadinovirus strain 17577 (RRV strain 17577) genome is essentially colinear with human herpesvirus 8 (HHV8)/Kaposi’s sarcoma-associated herpesvirus (KSHV) and encodes several analogous open reading frames (ORFs), including the homologue of cellular interleukin-6 (IL-6). To determine if the RRV IL-6-like ORF (RvIL-6) is biologically functional, it was expressed either transiently in COS-1 cells or purified from bacteria as a glutathioneS-transferase (GST)-RvIL-6 fusion and analyzed by IL-6 bioassays. Utilizing the IL-6-dependent B9 cell line, we found that both forms of RvIL-6 supported cell proliferation in a dose-dependent manner. Moreover, antibodies specific to the IL-6 receptor (IL-6R) or the gp130 subunit were capable of blocking the stimulatory effects of RvIL-6. Reciprocal titrations of GST-RvIL-6 against human recombinant IL-6 produced a more-than-additive stimulatory effect, suggesting that RvIL-6 does not inhibit but may instead potentiate normal cellular IL-6 signaling to B cells. These results demonstrate that RRV encodes an accessory protein with IL-6-like activity.

scholarly journals Intracellular Localization Map of Human Herpesvirus 8 Proteins

2007 ◽  
Vol 82 (4) ◽  
pp. 1908-1922 ◽  
Author(s):  
Gaby Sander ◽  
Andreas Konrad ◽  
Mathias Thurau ◽  
Effi Wies ◽  
Rene Leubert ◽  
...  
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Human Herpesvirus ◽  
Open Reading Frames ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Binding Partners ◽  
Infected Cells ◽  
Intracellular Translocation

ABSTRACT Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 K-bZIP Modulates Latency-Associated Nuclear Protein-Mediated Suppression of Lytic Origin-Dependent DNA Synthesis

2009 ◽  
Vol 83 (17) ◽  
pp. 8492-8501 ◽  
Author(s):  
Cyprian Rossetto ◽  
Irena Yamboliev ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Protein ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Dependent Manner ◽  
Herpesvirus 8 ◽  
Replication Assay ◽  
Lytic Dna Replication

ABSTRACT The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis. We developed a modification of the transient cotransfection replication assay wherein both lytic (oriLyt) and latent (terminal repeat) DNA replication are evaluated simultaneously. We now show that LANA represses origin-dependent lytic DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression was overcome by increasing amounts of a K-bZIP expression plasmid in the cotransfection mixture or by dominant-negative inhibition of the interaction of LANA with K-bZIP by the overexpression of the K-bZIP-LANA binding domain. Chromatin immunoprecipitation assays show that LANA interacts with oriLyt in the absence of K-bZIP expression, suggesting that suppression of lytic replication by LANA is mediated by direct binding. The interaction of K-bZIP with oriLyt was dependent upon the expression of LANA; however, LANA interacted with oriLyt independently of K-bZIP expression. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication and that K-bZIP facilitates lytic DNA replication and modulates the switch from the latent phase of the virus.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Two Functional Lytic Origins of DNA Replication

2002 ◽  
Vol 76 (15) ◽  
pp. 7890-7896 ◽  
Author(s):  
David P. AuCoin ◽  
Kelly S. Colletti ◽  
Yiyang Xu ◽  
Sylvia A. Cei ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Reading Frame ◽  
Herpesvirus 8 ◽  
Replication Assay ◽  
Inverted Duplication ◽  
Origin Of Dna Replication ◽  
The Right

ABSTRACT We used a transient-transfection replication assay to identify two functional copies of the human herpesvirus 8 (HHV8) lytic origin of DNA replication (oriLyt). BCLB-1 cells were transfected with HHV8 subgenomic fragments containing the putative lytic origin along with a plasmid expressing viral transactivator open reading frame (ORF) 50. The HHV8 left-end oriLyt (oriLyt-L) lies between ORFs K4.2 and K5 and is composed of a region encoding various transcription factor binding sites and an A+T-rich region and a G+C repeat region. The right-end oriLyt (oriLyt-R) maps between ORF 69 and vFLIP, a region similar to the RRV oriLyt, and is an inverted duplication of oriLyt-L.

scholarly journals Isolation and expression of three open reading frames from ovine herpesvirus-2

2002 ◽  
Vol 83 (3) ◽  
pp. 533-543 ◽  
Author(s):  
Lesley J. Coulter ◽  
Hugh W. Reid
Keyword(s):  
Human Herpesvirus ◽  
Open Reading Frames ◽  
Nuclear Antigen ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Lytic Gene ◽  
Herpesvirus 8 ◽  
Processivity Factor ◽  
Herpesvirus 1 ◽  
Reading Frames

Ovine herpesvirus-2 (OvHV-2), a member of the gammaherpesviruses (genus Rhadinovirus), asymptomatically infects its natural host, the sheep, but causes malignant catarrhal fever (MCF) in susceptible hosts, such as cattle, deer and pigs. A permissive cell culture system for virus replication has not been identified but viral DNA is present within lymphoblastoid cell lines (LCLs) established from cases of MCF. During this study, a cDNA expression library generated from LCLs was screened with sheep sera and two cDNAs were isolated. One cDNA contained two open reading frames (ORFs) that show similarity to ORFs 58 and 59 of alcelaphine herpesvirus-1 (AlHV-1), a closely related gammaherpesvirus that also causes MCF. Both ORFs 58 and 59 are conserved throughout the gammaherpesviruses. ORF 58 is predicted to be a membrane protein, while ORF 59 has been shown to be an early lytic gene that functions as a DNA polymerase processivity factor. The second cDNA clone contained a partial ORF showing limited similarity to AlHV-1 ORF 73, a homologue of the latency-associated nuclear antigen of human herpesvirus-8, which is associated with latent infections. The full-length OvHV-2 ORF 73 was cloned subsequently by PCR. The ORFs isolated from the library were cloned into a bacterial expression vector and the recombinant proteins tested for their reactivity to sera from OvHV-2-infected animals. An ORF 59 fusion protein was recognized specifically by sera from OvHV-2-infected cattle and will be used to develop a sero-diagnostic test.

scholarly journals Lipid Rafts of Primary Endothelial Cells Are Essential for Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8-Induced Phosphatidylinositol 3-Kinase and RhoA-GTPases Critical for Microtubule Dynamics and Nuclear Delivery of Viral DNA but Dispensable for Binding and Entry

2007 ◽  
Vol 81 (15) ◽  
pp. 7941-7959 ◽  
Author(s):  
Hari Raghu ◽  
Neelam Sharma-Walia ◽  
Mohanan Valiya Veettil ◽  
Sathish Sadagopan ◽  
Adriana Caballero ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Phosphatidylinositol 3 Kinase ◽  
Viral Dna ◽  
Herpesvirus 8 ◽  
Kshv Infection ◽  
Rhoa Gtpase ◽  
D Cells

ABSTRACT Early during de novo infection of human microvascular dermal endothelial (HMVEC-d) cells, Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8 [HHV-8]) induces the host cell's preexisting FAK, Src, phosphatidylinositol 3-kinase (PI3-K), Rho-GTPases, Diaphanous-2 (Dia-2), Ezrin, protein kinase C-ζ, extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB signal pathways that are critical for virus entry, nuclear delivery of viral DNA, and initiation of viral gene expression. Since several of these signal molecules are known to be associated with lipid raft (LR) domains, we investigated the role of LR during KSHV infection of HMVEC-d cells. Pretreatment of cells with LR-disrupting agents methyl β-cyclo dextrin (MβCD) or nystatin significantly inhibited the expression of viral latent (ORF73) and lytic (ORF50) genes. LR disruption did not affect KSHV binding but increased viral DNA internalization. In contrast, association of internalized viral capsids with microtubules (MTs) and the quantity of infected nucleus-associated viral DNA were significantly reduced. Disorganized and disrupted MTs and thick rounded plasma membranes were observed in MβCD-treated cells. LR disruption did not affect KSHV-induced FAK and ERK1/2 phosphorylation; in contrast, it increased the phosphorylation of Src, significantly reduced the KSHV-induced PI3-K and RhoA-GTPase and NF-κB activation, and reduced the colocalizations of PI3-K and RhoA-GTPase with LRs. Biochemical characterization demonstrated the association of activated PI3-K with LR fractions which was inhibited by MβCD treatment. RhoA-GTPase activation was inhibited by PI3-K inhibitors, demonstrating that PI3-K is upstream to RhoA-GTPase. In addition, colocalization of Dia-2, a RhoA-GTPase activated molecule involved in MT activation, with LR was reduced. KSHV-RhoA-GTPase mediated acetylation and aggregation of MTs were also reduced. Taken together, these studies suggest that LRs of endothelial cells play critical roles in KSHV infection and gene expression, probably due to their roles in modulating KSHV-induced PI3-K, RhoA-GTPase, and Dia-2 molecules essential for postbinding and entry stages of infection such as modulation of microtubular dynamics, movement of virus in the cytoplasm, and nuclear delivery of viral DNA.

scholarly journals The Immunogenic Glycoprotein gp35-37 of Human Herpesvirus 8 Is Encoded by Open Reading Frame K8.1

1998 ◽  
Vol 72 (8) ◽  
pp. 6725-6731 ◽  
Author(s):  
Marc-Steffen Raab ◽  
Jens-Christian Albrecht ◽  
Alexander Birkmann ◽  
Svenja Yağuboğlu ◽  
Dieter Lang ◽  
...  
Keyword(s):  
Phorbol Esters ◽  
Genomic Sequence ◽  
Human Herpesvirus ◽  
Body Cavity ◽  
Lytic Replication ◽  
Open Reading Frames ◽  
Human Herpesvirus 8 ◽  
Western Blots ◽  
Reading Frame ◽  
Herpesvirus 8

ABSTRACT Human herpesvirus 8 (HHV-8) is likely to be involved in the pathogenesis of Kaposi’s sarcoma (KS) and body cavity-based lymphomas (BCBLs). The HHV-8 genome is primarily in a latent state in BCBL-derived cell lines like BCBL-1, but lytic replication can be induced by phorbol esters (R. Renne, W. Zhang, B. Herndier, M. McGrath, N. Abbey, D. Kedes, and D. E. Ganem, Nat. Med. 2:342–346, 1996). A 35- to 37-kDa glycoprotein (gp35-37) is the polypeptide most frequently and intensively recognized by KS patient sera on Western blots with induced BCBL-1 cells. Its apparent molecular mass is reduced to 30 kDa by digestion with peptide-N-glycosidase F. By searching the known HHV-8 genomic sequence for open reading frames (ORF) with the potential to encode such a glycoprotein, an additional, HHV-8-specific reading frame was identified adjacently to ORF K8. This ORF, termed K8.1, was found to be transcribed primarily into a spliced mRNA encoding a glycoprotein of 228 amino acids. Recombinant K8.1 was regularly recognized by KS patient sera in Western blots, and immunoaffinity-purified antibodies to recombinant K8.1 reacted with gp35-37. This shows that the immunogenic gp35-37 is encoded by HHV-8 reading frame K8.1, which will be a useful tool for studies of HHV-8 epidemiology and pathogenesis.

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