Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

The oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus, also identified as human herpesvirus 8, contains genes producing proteins that control transcription and influence cell signaling. Open reading frame 36 (ORF36) of this virus encodes a serine/threonine protein kinase, which is designated the viral protein kinase (vPK). Our recent efforts to elucidate the role of vPK in the viral life cycle have focused on identifying viral protein substrates and determining the effects of vPK-mediated phosphorylation on specific steps in viral replication. The vPK gene was transcribed into 4.2-kb and 3.6-kb mRNAs during the early and late phases of viral reactivation. vPK is colocalized with viral DNA replication/transcription compartments as marked by a polymerase processivity factor, and K-bZIP, a protein known to bind the viral DNA replication origin (Ori-Lyt) and to regulate viral transcription. The vPK physically associated with and strongly phosphorylated K-bZIP at threonine 111, a site also recognized by the cyclin-dependent kinase Cdk2. Both K-bZIP and vPK were corecruited to viral promoters targeted by K-bZIP as well as to the Ori-Lyt region. Phosphorylation of K-bZIP by vPK had a negative impact on K-bZIP transcription repression activity. The extent of posttranslational modification of K-bZIP by sumoylation, a process that influences its repression function, was decreased by vPK phosphorylation at threonine 111. Our data thus identify a new role of vPK as a modulator of viral transcription.Kaposi's sarcoma-associated herpesvirus (KSHV), also designated human herpesvirus 8, has been linked to several malignancies, including Kaposi's sarcoma, B-cell lymphomas, primary effusion lymphomas, and multicentric Castleman's disease in immunocompromised individuals (reviewed in reference 46). Kaposi's sarcoma is the most common malignancy associated with AIDS (45). The viral genome is doublestranded DNA, approximately 165 kbp, and encodes over 81 open reading frames (ORFs) (43). The majority of the ORFs are essential for viral replication and include genes necessary for viral DNA replication, transcription, and assembly of infectious particles (51). In addition, the KSHV genome contains a large number of ORFs with homology to known cellular genes. Several of these viral ORFs are implicated in modulating host immune responses, promoting angiogenesis, and dysregulating cell growth (14). A model for regulated expression of KSHV genes has been deduced from numerous genetic and biochemical studies of viral RNA patterns and activities of viral transactivators (22,31,39,49). As for other herpesviruses, KSHV genes in productive infection have been classified into the following temporally distinct classes: immediate-early, early, and late. This virus can also establish a latent state that is characterized by presence of a multicopy circular episome of viral DNA, which expresses a small subset of viral proteins. Productive (lytic) viral replication can be induced by treatment of latently infected cell lines with butyrate,...

Section: Discussionmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

Izumiya

Geelen

et al. 2007

“…These ori-Lyts are located in the KSHV genome between K4.2 and K5 and between K12 and ORF71, respectively. These two ori-Lyts share almost identical 1.1-kb core component sequences and 600-bp GCrich repeats, which are represented as 20-bp and 30-bp tandem arrays (25).Like that of other herpesviruses, KSHV lytic DNA replication relies on virus-encoded proteins (2,38,41). Six core replication proteins, including a DNA polymerase (POL), a polymerase processivity factor (PPF), a single-stranded DNA binding protein (SSB), and a trivalent helicase-primase complex (HEL, PRI, and PAP) have been identified in the KSHV genome.…”

mentioning

confidence: 99%

“…Like that of other herpesviruses, KSHV lytic DNA replication relies on virus-encoded proteins (2,38,41). Six core replication proteins, including a DNA polymerase (POL), a polymerase processivity factor (PPF), a single-stranded DNA binding protein (SSB), and a trivalent helicase-primase complex (HEL, PRI, and PAP) have been identified in the KSHV genome.…”

mentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus ori - Lyt -Dependent DNA Replication: Involvement of Host Cellular Factors

Wang

Tang

et al. 2008

111

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is the etiologic agent of several AIDS-associated malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease (1,17,33). In KS lesions, most spindle cells of endothelial origin are latently infected with KSHV, and a small percentage of these cells undergo spontaneous lytic replication (34,35,43). Increasing evidence suggests that the small percentage of cells experiencing viral lytic replication play important roles in viral pathogenicity. Treatment of KS patients or AIDS patients at risk for KS with antiherpesviral drugs, like foscarnet and ganciclovir, which are known to block lytic DNA replication, results in regression of KS lesions or a decease in the incidence of KS development. The lytic replication cycle directly contributes to viral tumorigenesis by spreading viruses to target cells and by providing paracrine regulation for KS development (11). A recent study by Grundhoff and Ganem (19) suggests a new role for lytic replication in sustaining the population of latently infected cells that otherwise would be quickly lost by segregation of latent viral episomes as spindle cells divide. Thus, unlike with other oncogenic viruses, where the latent life c...

“…The K8.1 gene deletion spanned the K8.1 ORF immediately after the initiation codon (ATG) and extended to approximately 90 bases upstream of the K8.1 termination codon. KSHV ORF50, specifying the major lytic transactivator RTA, and the K8 ORF specifying K-bZIP, which has been suggested to act either as a repressor of lytic replication (23,28) or as a replication activator protein (59), lie immediately 5Ј to the K8.1 gene. All three genes code for mRNAs that utilize the same polyadenylation signal immediately downstream of K8.1.…”

mentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein K8.1 Is Dispensable for Virus Entry

Luna

Zhou

Baghian³

et al. 2004