A Novel Protein Encoded by Kaposi’s Sarcoma-Associated Herpesvirus Open Reading Frame 36 Inhibits Cell Spreading and Focal Adhesion Kinase Activation

Intervirology ◽  
2007 ◽  
Vol 50 (6) ◽  
pp. 426-432 ◽  
Author(s):  
Junsoo Park ◽  
Myung-Shin Lee ◽  
Seung-Min Yoo ◽  
Taegun Seo
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cell Spreading ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Kinase Activation ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Novel Protein

scholarly journals Structure of the Open Reading Frame 49 Protein Encoded by Kaposi's Sarcoma-Associated Herpesvirus

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Kelly Hew ◽  
Saranya Veerappan ◽  
Daniel Sim ◽  
Tobias Cornvik ◽  
Pär Nordlund ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Life Cycle ◽  
Dna Binding ◽  
Protein Interaction ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in Kaposi's sarcoma-associated herpesvirus (KSHV). The protein encoded by open reading frame 49 from KSHV (ORF49KSHV) has been shown to upregulate lytic replication in KSHV by enhancing the activities of the RTA. We have solved the crystal structure of the ORF49KSHV protein to a resolution of 2.4 Å. The ORF49KSHV protein has a novel fold consisting of 12 alpha-helices bundled into two pseudodomains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of the ORF49KSHV protein in the gammaherpesvirus subfamily have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions, suggesting that they are more likely to play important structural roles than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. The ORF49KSHV protein is stabilized in a dose-responsive manner by double-stranded oligonucleotides, suggesting actual DNA interaction and binding. Biolayer interferometry studies also demonstrated that the ORF49KSHV protein binds these oligonucleotides. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a tumorigenic gammaherpesvirus that causes multiple cancers and lymphoproliferative diseases. The virus exists mainly in the quiescent latent life cycle, but when it is reactivated into the lytic life cycle, new viruses are produced and disease symptoms usually manifest. Several KSHV proteins play important roles in this reactivation, but their exact roles are still largely unknown. In this study, we report the crystal structure of the open reading frame 49 protein encoded by KSHV (ORF49KSHV). Possible regions for protein interaction that could harbor functional importance were found on the surface of the ORF49KSHV protein. This led to the discovery of novel DNA binding properties of the ORF49KSHV protein. Evolutionary conserved structural elements with the functional homologs of ORF49KSHV were also established with the structure.

scholarly journals Focal Adhesion Kinase Is Critical for Entry of Kaposi's Sarcoma-Associated Herpesvirus into Target Cells

2006 ◽  
Vol 80 (3) ◽  
pp. 1167-1180 ◽  
Author(s):  
Harinivas H. Krishnan ◽  
Neelam Sharma-Walia ◽  
Daniel N. Streblow ◽  
Pramod P. Naranatt ◽  
Bala Chandran
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Viral Entry ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Target Cells ◽  
Viral Dna ◽  
Kshv Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface α3β1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK−/−) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK−/−) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities

2000 ◽  
Vol 74 (8) ◽  
pp. 3586-3597 ◽  
Author(s):  
Jessica R. Kirshner ◽  
David M. Lukac ◽  
Jean Chang ◽  
Don Ganem
Keyword(s):  
Posttranscriptional Regulation ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frame ◽  
Luciferase Reporter ◽  
Reading Frame ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Coding Potential

ABSTRACT Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.

scholarly journals Identification of Kaposin (Open Reading Frame K12) as a Human Herpesvirus 8 (Kaposi’s Sarcoma-Associated Herpesvirus) Transforming Gene

1998 ◽  
Vol 72 (6) ◽  
pp. 4980-4988 ◽  
Author(s):  
Sumitra Muralidhar ◽  
Anne M. Pumfery ◽  
Morad Hassani ◽  
M. Reza Sadaie ◽  
Norio Azumi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Body Cavity ◽  
Open Reading Frame ◽  
Human Herpesvirus 8 ◽  
Reading Frame ◽  
Herpesvirus 8 ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The recently identified human herpesvirus 8 (HHV-8, or Kaposi’s sarcoma-associated herpesvirus) has been implicated in the etiology of both Kaposi’s sarcoma (KS) and primary effusion (body cavity-based) lymphoma (PEL) (Y. Chang et al., Science 266:1865–1869, 1994; P. S. Moore et al., J. Virol. 70:549–558, 1996). An important feature of the association of HHV-8 with these malignancies is the expression of an abundant, latency-associated 0.7-kb transcript, T0.7 (W. Zhong et al., Proc. Natl. Acad. Sci. USA 93:6641–6646, 1996). T0.7 is found in all stages in nearly all KS tumors of different epidemiologic origin, including AIDS-associated, African endemic, and classical KS (K. A. Staskus et al., J. Virol. 71:715–719, 1997), as well as in a body cavity-based lymphoma-derived cell line, BCBL-1, that is latently infected with HHV-8 (R. Renne et al., Nat. Med. 2:342–346, 1996). T0.7 encodes a unique HHV-8 open reading frame, K12, also known as kaposin. In this study, we report that the kaposin gene induced tumorigenic transformation. Constructs with kaposin expressed either from its endogenous promoter or from a heterologous promoter induced focal transformation upon transfection into Rat-3 cells. All transformed Rat-3 cell lines containing kaposin sequences produced high-grade, highly vascular, undifferentiated sarcomas upon subcutaneous injection of athymic nu/nu mice. Tumor-derived cell lines expressed kaposin mRNA, suggesting a role in the maintenance of the transformed phenotype. Furthermore, kaposin protein was detected in transformed and tumor-derived cells by immunofluorescence and localized to the cytoplasm. More importantly, expression of kaposin protein was also detected in the PEL cell lines BCBL-1 and KS-1. These findings demonstrate the oncogenic potential of kaposin and suggest its possible role in the development of KS and other HHV-8-associated malignancies.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50/Rta Protein Activates the Entire Viral Lytic Cycle in the HH-B2 Primary Effusion Lymphoma Cell Line

2000 ◽  
Vol 74 (13) ◽  
pp. 6207-6212 ◽  
Author(s):  
Lyndle Gradoville ◽  
Jennifer Gerlach ◽  
Elizabeth Grogan ◽  
Duane Shedd ◽  
Sarah Nikiforow ◽  
...  
Keyword(s):  
Cell Line ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frame ◽  
Lytic Cycle ◽  
Viral Dna ◽  
Reading Frame ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.

scholarly journals An Sp1 Response Element in the Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50 Promoter Mediates Lytic Cycle Induction by Butyrate

2005 ◽  
Vol 79 (3) ◽  
pp. 1397-1408 ◽  
Author(s):  
Jianjiang Ye ◽  
Duane Shedd ◽  
George Miller
Keyword(s):  
Sodium Butyrate ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frame ◽  
Lytic Cycle ◽  
Response Element ◽  
Reading Frame ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) can be driven into the lytic cycle in vitro by phorbol esters and sodium butyrate. This report begins to analyze the process by which butyrate activates the promoter of KSHV open reading frame 50 (ORF50), the key viral regulator of the KSHV latency to lytic cycle switch. A short fragment of the promoter, 134 nucleotides upstream of the translational start of ORF50, retained basal uninduced activity and conferred maximal responsiveness to sodium butyrate. The butyrate response element was mapped to a consensus Sp1-binding site. By means of electrophoretic mobility shift assays, both Sp1 and Sp3 were shown to form complexes in vitro with the ORF50 promoter at the Sp1 site. Butyrate induced the formation of a group of novel complexes, including several Sp3-containing complexes, one Sp1-containing complex, and several other complexes that were not identified with antibodies to Sp1 or Sp3. Formation of all butyrate-induced DNA-protein complexes was mediated by the consensus Sp1 site. In insect and mammalian cell lines, Sp1 significantly activated the ORF50 promoter linked to luciferase. Chromatin immunoprecipitation experiments in a PEL cell line showed that butyrate induced Sp1, CBP, and p300 binding to the ORF50 promoter in vivo in an on-off manner. The results suggest that induction of the KSHV lytic cycle by butyrate is mediated through interactions at the Sp1/Sp3 site located 103 to 112 nucleotides upstream of the translational initiation of ORF50 presumably by enhancing the binding of Sp1 to this site.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Open Reading Frame 4 Protein (Kaposica) Is a Functional Homolog of Complement Control Proteins

2003 ◽  
Vol 77 (6) ◽  
pp. 3878-3881 ◽  
Author(s):  
Jayati Mullick ◽  
John Bernet ◽  
Akhilesh K. Singh ◽  
John D. Lambris ◽  
Arvind Sahu
Keyword(s):  
Kaposi's Sarcoma ◽  
Expression System ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Complement Control Proteins ◽  
Functional Homolog ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.

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