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Open Access Publications from the University of California

Scholarly Works (39 results)

  • Article
  • Peer Reviewed
UC Davis Previously Published Works (2020)
Kaposi's sarcoma-associated herpesvirus (KSHV), also designated human herpesvirus 8 (HHV-8), has been linked to Kaposi's sarcoma, as well as to primary effusion lymphoma (PEL), and a subset of multicentric Castleman's disease. KSHV genomes are maintained as episomes within infected cells and the virus exhibits a biphasic life cycle consisting of a life-long latent phase during which only a few viral genes are expressed and no viral progeny are produced and a transient lytic reactivation phase, in which a full repertoire of ~ 80 lytic genes are activated in a temporally regulated manner culminating in the release of new virions. Lytic replication is initiated by a single viral protein, K-Rta (ORF50), which activates more than 80 viral genes from multiple resident viral episomes (i.e., viral chromosomes). One of the major targets of K-Rta is a long non-coding nuclear RNA, PAN RNA (polyadenylated nuclear RNA), a lncRNA that accumulates to exceedingly high levels in the nucleus during viral reactivation. K-Rta directly binds to the PAN RNA promoter and robustly activates PAN RNA expression. Although PAN RNA has been known for over 20 years, its role in viral replication is still incompletely understood. In this perspective, we will briefly review the current understanding of PAN RNA and then describe our current working model of this RNA. The model is based on our observations concerning events that occur during KSHV lytic reactivation including (i) a marked accumulation of RNA Pol II at the PAN promoter, (ii) genomic looping emanating from the PAN locus, (iii) interaction of a second viral lytic protein (ORF57) with K-Rta, PAN RNA and RNA Pol II, (iv) the essential requirement for PAN RNA expression in cis for optimal transcriptional execution needed for the entire lytic program, and (v) ORF57 recruitment of RNA Pol II to the PAN genomic locus. Together our results generate a model in which the PAN locus serves as a hub for sequestration/trapping of the cellular transcriptional machinery proximal to viral episomes. Sequestration at the PAN locus facilitates high levels of viral transcription throughout the viral genome during lytic replication. ORF57 acts as a transcription-dependent transactivator at the PAN locus by binding to both Rta and PAN to locally trap RNA Pol II. The resulting accumulation of high levels of nuclear PAN RNA created by this process is an inducible enhancer-derived (eRNA) by-product that litters the infected cell nucleus.
    Cover page: PAN RNA: transcriptional exhaust from a viral engine
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2012)
    KSHV latency can be envisioned as an outcome that is balanced between factors that promote viral gene expression and lytic replication against those that facilitate gene silencing and establish or maintain latency. A large body of work has focused on the activities of the key viral regulatory proteins involved in KSHV latent or lytic states. Moreover, recent studies have also begun to document the importance of epigenetic landscape evolution of the KSHV viral genome during latency and reactivation. However, one area of KSHV molecular virology that remains largely unanswered is the precise role of post-translational modifications on the activities of viral factors that function during latency and reactivation. In this review, we will summarize the post-translational modifications associated with three viral factors whose activities contribute to the viral state. The viral proteins discussed are the two major KSHV encoded transcription factors, K-Rta (KSHV replication and transcriptional activator) and K-bZIP (KSHV basic leucine zipper) and the viral latency-associated nuclear antigen (LANA). A special emphasis will be placed on the role of the sumoylation pathway in the modulation of the KSHV lifecycle. Newly uncovered small ubiquitin-like modifier (SUMO)-associated properties of LANA and K-Rta will also be presented, namely LANA histone targeting SUMO E3 ligase activity and K-Rta SUMO-targeted ubiquitin ligase function.
      Cover page: Post-Translational Modifications of Kaposi’s Sarcoma-Associated Herpesvirus Regulatory Proteins – SUMO and KSHV
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2020)
      Kaposi's sarcoma-associated herpesvirus (KSHV), also designated human herpesvirus 8 (HHV-8), has been linked to Kaposi's sarcoma, as well as to primary effusion lymphoma (PEL), and a subset of multicentric Castleman's disease. KSHV genomes are maintained as episomes within infected cells and the virus exhibits a biphasic life cycle consisting of a life-long latent phase during which only a few viral genes are expressed and no viral progeny are produced and a transient lytic reactivation phase, in which a full repertoire of ~ 80 lytic genes are activated in a temporally regulated manner culminating in the release of new virions. Lytic replication is initiated by a single viral protein, K-Rta (ORF50), which activates more than 80 viral genes from multiple resident viral episomes (i.e., viral chromosomes). One of the major targets of K-Rta is a long non-coding nuclear RNA, PAN RNA (polyadenylated nuclear RNA), a lncRNA that accumulates to exceedingly high levels in the nucleus during viral reactivation. K-Rta directly binds to the PAN RNA promoter and robustly activates PAN RNA expression. Although PAN RNA has been known for over 20 years, its role in viral replication is still incompletely understood. In this perspective, we will briefly review the current understanding of PAN RNA and then describe our current working model of this RNA. The model is based on our observations concerning events that occur during KSHV lytic reactivation including (i) a marked accumulation of RNA Pol II at the PAN promoter, (ii) genomic looping emanating from the PAN locus, (iii) interaction of a second viral lytic protein (ORF57) with K-Rta, PAN RNA and RNA Pol II, (iv) the essential requirement for PAN RNA expression in cis for optimal transcriptional execution needed for the entire lytic program, and (v) ORF57 recruitment of RNA Pol II to the PAN genomic locus. Together our results generate a model in which the PAN locus serves as a hub for sequestration/trapping of the cellular transcriptional machinery proximal to viral episomes. Sequestration at the PAN locus facilitates high levels of viral transcription throughout the viral genome during lytic replication. ORF57 acts as a transcription-dependent transactivator at the PAN locus by binding to both Rta and PAN to locally trap RNA Pol II. The resulting accumulation of high levels of nuclear PAN RNA created by this process is an inducible enhancer-derived (eRNA) by-product that litters the infected cell nucleus.
        Cover page: PAN RNA: transcriptional exhaust from a viral engine
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2018)
        It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.
          Cover page: Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy.
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2014)
          Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is a γ-herpesvirus linked to Kaposi's sarcoma (KS) and two lymphoproliferative disorders, primary effusion lymphoma (PEL or body-cavity B-lymphoma [BCBL]) and a subset of Multicentric Castleman's Disease. During lytic growth, pervasive viral transcription generating a variety of transcripts with uncertain protein-coding potential has been described on a genome-wide scale in β- and γ-herpesviruses. One class of such RNAs is called long non-coding RNAs (lncRNAs). KSHV encodes a viral lncRNA known as polyadenylated nuclear RNA (PAN RNA), a copious early gene product. PAN RNA has been implicated in KSHV gene expression, replication, and immune modulation. PAN RNA expression is required for optimal expression of the entire KSHV lytic gene expression program. Latent KSHV episomes are coated with viral latency-associated nuclear antigen (LANA). LANA rapidly dissociates from episomes during reactivation. Here we review recent studies suggesting that PAN RNA may function as a viral lncRNA, including a role in the facilitation of LANA-episomal dissociation during lytic replication.
            Cover page: Long Non-Coding RNA and Epigenetic Gene Regulation of KSHV
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2021)
            Marek's disease virus (MDV) encodes a basic-leucine zipper (BZIP) protein, Meq, which is considered the major MDV oncoprotein. It has been reported that the oncogenicity of Meq is associated with its interaction with C-terminal binding protein 1 (CtBP), which is also an interaction partner of Epstein-Barr virus encoded EBNA3A and EBNA3C oncoproteins. Since both EBNA3C and CtBP interact with histone deacetylase 1 (HDAC1) and HDAC2, we examined whether Meq shares this interaction with chicken HDAC1 (chHDAC1) and chHDAC2. Using confocal microscopy analysis, we show that Meq co-localizes with chHDAC1 and chHDAC2 in the nuclei of MDV lymphoblastoid tumor cells. In addition, immunoprecipitation assays demonstrate that Meq interacts with chHDAC1 and chHDAC2 in transfected cells and MDV lymphoblastoid tumor cells. Using deletion mutants, interaction domains were mapped to the N-terminal dimerization domain of chHDAC1 and chHDAC2, and the BZIP domain of Meq. Our results further demonstrate that this interaction mediates the degradation of chHDAC1 and chHDAC2 via the proteasome dependent pathway. In addition, our results show that Meq also induces the reduction of global ubiquitinated proteins through a proteasome dependent pathway. In conclusion, our results provide evidence that Meq interacts with chHDAC1 and chHDAC2, and induces their proteasome dependent degradation.
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2014)
              The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvβ3 predicted that mCRP binds to αvβ3 well. We found that mCRP actually bound to integrins αvβ3 and α4β1 well. Antagonists to αvβ3 or α4β1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin β1 mutant (β1-3-1) that has a small fragment from the ligand binding site of β3, we showed that mCRP bound to the classical RGD-binding site in αvβ3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvβ3 and α4β1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvβ3 and α4β1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvβ3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.
                Cover page: The Binding of Monomeric C-Reactive Protein (mCRP) to Integrins αvβ3 and α4β1 Is Related to Its Pro-Inflammatory Action
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2024)
                Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.
                  Cover page: Kaposi’s sarcoma-associated herpesvirus (KSHV) LANA prevents KSHV episomes from degradation
                  • Article
                  • Peer Reviewed
                  California HIV/AIDS Research Program Funded Publications (2009)

                  Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma. K-Rta and K-bZIP are two major viral transcription factors that control reactivation of this virus. Here we report a genome-wide analysis of transcriptional capacity by evaluation of a comprehensive library of 83 putative KSHV promoters. In reporter assays, 34 viral promoters were activated by K-Rta, whereas K-bZIP activated 21 promoters. When K-Rta and K-bZIP were combined, 3 K-Rta responsive promoters were repressed by K-bZIP. The occupancy of K-Rta and K-bZIP across KSHV promoters was analyzed by chromatin immunoprecipitation with a viral promoter-chip in BCBL-1 cells. In addition, acetylation of local histones was examined to determine accessibility of promoters during latency and reactivation. Finally, 10 promoters were selected to study the dynamics of transcription factor recruitment. This study provides a comprehensive overview of the responsiveness of KSHV promoters to K-Rta and K-bZIP, and describes key chromatin changes during viral reactivation.

                    Cover page: A comprehensive analysis of recruitment and transactivation potential of K-Rta and K-bZIP during reactivation of Kaposi's sarcoma-associated herpesvirus.
                    • Article
                    • Peer Reviewed
                    California HIV/AIDS Research Program Funded Publications (2009)

                    Successful viral replication is dependent on a conducive cellular environment; thus, viruses must be sensitive to the state of their host cells. We examined the idea that an interplay between viral and cellular regulatory factors determines the switch from Kaposi's sarcoma-associated herpesvirus (KSHV) latency to lytic replication. The immediate-early gene product K-Rta is the first viral protein expressed and an essential factor in reactivation; accordingly, this viral protein is in a key position to serve as a viral sensor of cellular physiology. Our approach aimed to define a host transcription factor, i.e., host sensor, which modulates K-Rta activity on viral promoters. To this end, we developed a panel of reporter plasmids containing all 83 putative viral promoters for a comprehensive survey of the response to both K-Rta and cellular transcription factors. Interestingly, members of the NF-kappaB family were shown to be strong negative regulators of K-Rta transactivation for all but two viral promoters (Ori-RNA and K12). Recruitment of K-Rta to the ORF57 and K-bZIP promoters, but not the K12 promoter, was significantly impaired when NF-kappaB expression was induced. Many K-Rta-responsive promoters modulated by NF-kappaB contain the sequence of the RBP-Jkappa binding site, a major coactivator which anchors K-Rta to target promoters via consensus motifs which overlap with that of NF-kappaB. Gel shift assays demonstrated that NF-kappaB inhibits the binding of RBP-Jkappa and forms a complex with RBP-Jkappa. Our results support a model in which a balance between K-Rta/RBP-Jkappa and NF-kappaB activities determines KSHV reactivation. An important feature of this model is that the interplay between RBP-Jkappa and NF-kappaB on viral promoters controls viral gene expression mediated by K-Rta.

                      Cover page: NF-kappaB serves as a cellular sensor of Kaposi's sarcoma-associated herpesvirus latency and negatively regulates K-Rta by antagonizing the RBP-Jkappa coactivator.
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