scholarly journals Cloning and Functional Analysis of Kaposi’s Sarcoma-Associated Herpesvirus DNA Polymerase and Its Processivity Factor

1998 ◽  
Vol 72 (7) ◽  
pp. 6228-6232 ◽  
Author(s):  
Kai Lin ◽  
Charlotte Y. Dai ◽  
Robert P. Ricciardi
Keyword(s):  
Dna Polymerase ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Herpes Simplex Virus 1 ◽  
Processivity Factor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Simplex Virus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified virus with tumorigenic potential. Here, we cloned and expressed the DNA polymerase (Pol-8) of KSHV and its processivity factor (PF-8). Pol-8 bound specifically to PF-8 in vitro. Moreover, the DNA synthesis activity of Pol-8 was shown in vitro to be strongly dependent on PF-8. Addition of PF-8 to Pol-8 allowed efficient synthesis of fully extended DNA products corresponding to the full-length M13 template (7,249 nucleotides), whereas Pol-8 alone could incorporate only several nucleotides. The specificity of PF-8 and Pol-8 for each other was demonstrated by their inability to be functionally replaced by the DNA polymerases and processivity factors of herpes simplex virus 1 and human herpesvirus 6.

scholarly journals The Crystal Structure of PF-8, the DNA Polymerase Accessory Subunit from Kaposi's Sarcoma-Associated Herpesvirus

2009 ◽  
Vol 83 (23) ◽  
pp. 12215-12228 ◽  
Author(s):  
Jennifer L. Baltz ◽  
David J. Filman ◽  
Mihai Ciustea ◽  
Janice Elaine Y. Silverman ◽  
Catherine L. Lautenschlager ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Polymerase ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Catalytic Subunit ◽  
Accessory Subunit ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Simplex Virus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Related Proteins

ABSTRACT Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.8 Å. This structure reveals that each monomer of PF-8 shares a fold common to processivity factors. Like human cytomegalovirus UL44, PF-8 forms a head-to-head dimer in the form of a C clamp, with its concave face containing a number of basic residues that are predicted to be important for DNA binding. However, there are several differences with related proteins, especially in loops that extend from each monomer into the center of the C clamp and in the loops that connect the two subdomains of each protein, which may be important for determining PF-8's mode of binding to DNA and to Pol-8. Using the crystal structures of PF-8, the herpes simplex virus catalytic subunit, and RB69 bacteriophage DNA polymerase in complex with DNA and initial experiments testing the effects of inhibition of PF-8-stimulated DNA synthesis by peptides derived from Pol-8, we suggest a model for how PF-8 might form a ternary complex with Pol-8 and DNA. The structure and the model suggest interesting similarities and differences in how PF-8 functions relative to structurally similar proteins.

scholarly journals Potent Antiviral Activity of North-Methanocarbathymidine against Kaposi's Sarcoma-Associated Herpesvirus

2005 ◽  
Vol 49 (12) ◽  
pp. 4965-4973 ◽  
Author(s):  
Weimin Zhu ◽  
Angela Burnette ◽  
Dorjbal Dorjsuren ◽  
Paula E. Roberts ◽  
Mahmoud Huleihel ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Virion Production ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Simplex Virus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS). Blocking lytic KSHV replication may hinder KS tumorigenesis. Here, we report potent in vitro anti-KSHV activity of 2′-exo-methanocarbathymidine [North-methanocarbathymidine (N-MCT)], a thymidine analog with a pseudosugar ring locked in the northern conformation, which has previously been shown to block the replication of herpes simplex virus types 1 and 2. N-MCT inhibited KSHV virion production in lytically induced KSHV-infected BCBL-1 cells with a substantially lower 50% inhibitory concentration (IC50) than those of cidofovir (CDV) and ganciclovir (GCV) (IC50, mean ± standard deviation: 0.08 ± 0.03, 0.42 ± 0.07, and 0.96 ± 0.49 μM for N-MCT, CDV, and GCV, respectively). The reduction in KSHV virion production was accompanied by a corresponding decrease in KSHV DNA levels in the N-MCT-treated BCBL-1 cells, indicating that the compound blocked lytic KSHV DNA replication. A time- and dose-dependent accumulation of N-MCT-triphosphate (TP) was demonstrated in lytically induced BCBL-1 cells, while uninfected cells showed virtually no accumulation. The levels of N-MCT-TP were significantly decreased in the presence of 5′-ethynylthymidine, a potent inhibitor of herpesvirus thymidine kinase, resulting in the abrogation of anti-KSHV activity of N-MCT. N-MCT-TP more effectively blocked in vitro DNA synthesis by KSHV DNA polymerase with an IC50 of 6.24 ± 0.08 μM (mean ± standard deviation) compared to CDV-diphosphate (14.70 ±2.47 μM) or GCV-TP (24.59 ± 5.60 μM). Taken together, N-MCT is a highly potent and target-specific anti-KSHV agent which inhibits lytic KSHV DNA synthesis through its triphosphate metabolite produced in KSHV-infected cells expressing a virally encoded thymidine kinase.

scholarly journals Construction of an Infectious Rhesus Rhadinovirus Bacterial Artificial Chromosome for the Analysis of Kaposi's Sarcoma-Associated Herpesvirus-Related Disease Development

2007 ◽  
Vol 81 (6) ◽  
pp. 2957-2969 ◽  
Author(s):  
Ryan D. Estep ◽  
Michael F. Powers ◽  
Bonnie K. Yen ◽  
He Li ◽  
Scott W. Wong
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Artificial Chromosome ◽  
Wild Type ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV17577) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV17577, producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system.

scholarly journals Wide-Scale Use of Notch Signaling Factor CSL/RBP-Jκ in RTA-Mediated Activation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Genes

2009 ◽  
Vol 84 (3) ◽  
pp. 1334-1347 ◽  
Author(s):  
Linda M. Persson ◽  
Angus C. Wilson
Keyword(s):  
Notch Signaling ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Notch Signaling Pathway ◽  
Lytic Replication ◽  
Reading Frame ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT For Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8 [HHV8]), the switch from latency to active lytic replication requires RTA, the product of open reading frame 50 (ORF50). RTA activates transcription from nearly 40 early and delayed-early viral promoters, mainly through interactions with cellular DNA binding proteins, such as CSL/RBP-Jκ, Oct-1, C/EBPα, and c-Jun. Reliance on cellular coregulators may allow KSHV to adjust its lytic program to suit different cellular contexts or interpret signals from the outside. CSL is a key component of the Notch signaling pathway and is targeted by several viruses. A search with known CSL binding sequences from cellular genes found at least 260 matches in the KSHV genome, many from regions containing known or suspected lytic promoters. Analysis of clustered sites located immediately upstream of ORF70 (thymidylate synthase), ORF19 (tegument protein), and ORF47 (glycoprotein L) uncovered RTA-responsive promoters that were validated using mRNAs isolated from KSHV-infected cells undergoing lytic reactivation. Notably, ORF19 behaves as a true late gene, indicating that RTA regulates all three phases of the lytic program. For each new promoter, the response to RTA was dependent on CSL, and 5 of the 10 candidate sites were shown to bind CSL in vitro. Analysis of individual sites highlighted the importance of a cytosine residue flanking the core CSL binding sequence. These findings broaden the role for CSL in coordinating the KSHV lytic gene expression program and help to define a signature motif for functional CSL sites within the viral genome.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Inhibits Lytic Replication by Targeting Rta: a Potential Mechanism for Virus-Mediated Control of Latency

2004 ◽  
Vol 78 (12) ◽  
pp. 6585-6594 ◽  
Author(s):  
Ke Lan ◽  
Daniel A. Kuppers ◽  
Subhash C. Verma ◽  
Erle S. Robertson
Keyword(s):  
Kaposi's Sarcoma ◽  
Critical Role ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Nuclear Antigen ◽  
Viral Progeny ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing

2007 ◽  
Vol 81 (15) ◽  
pp. 8225-8235 ◽  
Author(s):  
Hyun Jin Kwun ◽  
Suzane Ramos da Silva ◽  
Ishita M. Shah ◽  
Neil Blake ◽  
Patrick S. Moore ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses.

scholarly journals Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

2001 ◽  
Vol 75 (3) ◽  
pp. 1378-1386 ◽  
Author(s):  
Jeffrey Vieira ◽  
Patricia O'Hearn ◽  
Louise Kimball ◽  
Bala Chandran ◽  
Lawrence Corey
Keyword(s):  
Human Cytomegalovirus ◽  
Kaposi's Sarcoma ◽  
Human Fibroblasts ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) andneo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.

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