Architecture of replication compartments formed during Epstein-Barr virus lytic replication

Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.     

RSK1 SUMOylation is required for KSHV lytic replication

RSK1, a downstream kinase of the MAPK pathway, has been shown to regulate multiple cellular processes and is essential for lytic replication of a variety of viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. We demonstrate that RSK1 undergoes robust SUMOylation during KSHV lytic replication at lysine residues K110, K335, and K421. SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Compared to wild-type RSK1, the overall phosphorylation level of RxRxxS*/T* motif is significantly declined in RSK1^{K110/335/421R} expressing cells. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B. Sequence analysis showed that eIF4B has one SUMO-interacting motif (SIM) between the amino acid position 166 and 170 (₁₆₆IRVDV₁₇₀), which mediates the association between eIF4B and RSK1 through SUMO-SIM interaction. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication.     

EBV裂解复制周期调控机制研究新进展

EBV与许多恶性疾病包括霍奇金病、伯基特淋巴瘤、鼻咽癌等恶性肿瘤发病有关人类B淋巴细胞是EBV天然宿主,其在宿主细胞中的生活周期分为潜伏感染和裂解感染。EBV潜伏感染时,为逃避宿主细胞的免疫杀伤,仅表达少量基因产物。而在外界条件如化学、物理或宿主细胞分化的刺激下,EBV可由潜伏感染进入到裂解复制(Lytic Replication)感染周期,促进病毒在宿主细胞中播散。根据EBV裂解复制产物出现的时间顺序可将裂解复制周期分为裂解复制立即早期、早期和晚期。1 EBV裂解复制不同时期产物的调控作用     

Murine gammaherpesvirus 68 contains two functional lytic origins of replication

A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt.     

Activation of p90 ribosomal S6 kinase by ORF45 of Kaposi's sarcoma-associated herpesvirus and its role in viral lytic replication

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway is essential for infection by a variety of viruses. The p90 ribosomal S6 kinases (RSKs) are direct substrates of ERK and functional mediators of ERK MAPK signaling, but their roles in viral infection have never been examined. We demonstrate that ORF45 of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with RSK1 and RSK2 and strongly stimulates their kinase activities. The activation of RSK by ORF45 is correlated with ERK activation but does not require MEK. We further demonstrate that RSK1/RSK2 is activated during KSHV primary infection and reactivation from latency; a subset of RSK1/RSK2 is present in the viral replication compartment in the nucleus. Depletion of RSK1/RSK2 by small interfering RNA or the specific inhibitor BI-D1870 suppresses KSHV lytic gene expression and progeny virion production, suggesting an essential role of RSK1/RSK2 in KSHV lytic replication.     

The Epstein-Barr virus BMRF1 gene is essential for lytic virus replication

The Epstein-Barr virus (EBV) BMRF1 protein is a DNA polymerase processivity factor. We have deleted the BMRF1 open reading frame from the EBV genome and assessed the DeltaBMRF1 EBV phenotype. DeltaBMRF1 viruses were replication deficient, but the wild-type phenotype could be restored by BMRF1 trans-complementation. The replication-deficient phenotype included impaired lytic DNA replication and late protein expression. DeltaBMRF1 and wild-type viruses were undistinguishable in terms of their ability to transform primary B cells. Our results provide genetic evidence that BMRF1 is essential for lytic replication of the EBV genome.     

The SUMO E3 ligase activity of ORF45 determines KSHV lytic replication

RSK1, an essential cellular kinase for Kaposi’s sarcoma-associated herpesvirus (KSHV) replication, is highly phosphorylated and SUMOylated during KSHV lytic cycle, which determine the substrate phosphorylation and specificity of RSK1, respectively. However, the SUMO E3 ligase responsible for attaching SUMO to RSK1 has not yet been identified. By genome-wide screening, we found that KSHV ORF45 is necessary and sufficient to enhance RSK1 SUMOylation. Mechanistically, KSHV ORF45 binds to SUMOs via two classic SUMO-interacting motifs (SIMs) and functions as a SIM-dependent SUMO E3 ligase for RSK1. Mutations on these ORF45 SIMs resulted in much lower lytic gene expressions, viral DNA replication, and mature progeny virus production. Interestingly, KSHV ORF45 controls RSK1 SUMOylation and phosphorylation via two separated functional regions: SIMs and amino acid 17–90, respectively, which do not affect each other. Similar to KSHV ORF45, ORF45 of Rhesus Macaque Rhadinovirus has only one SIM and also increases RSK1 SUMOylation in a SIM-dependent manner, while other ORF45 homologues do not have this function. Our work characterized ORF45 as a novel virus encoded SUMO E3 ligase, which is required for ORF45-RSK1 axis-mediated KSHV lytic gene expression.     

Glycoprotein M is an essential lytic replication protein of the murine gammaherpesvirus 68

All herpesviruses encode a homolog of glycoprotein M (gM), which appears to function in virion morphogenesis. Despite its conservation, gM is inessential for the lytic replication of alphaherpesviruses. In order to address the importance of gM in gammaherpesviruses, we disrupted it in the murine gammaherpesvirus 68 (MHV-68). The mutant virus completely failed to propagate in normally permissive fibroblasts. The defective genome was rescued by either homologous recombination to restore the wild-type gM in situ or the insertion of an ectopic, intergenic expression cassette encoding gM into the viral genome. Thus, gM was essential for the lytic replication of MHV-68.     

Identification of cis sequences required for lytic DNA replication and packaging of murine gammaherpesvirus 68

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the cis elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (oriLyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated oriLyt plasmid into mature viral particles. Functional identification of the MHV-68 oriLyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.     

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 alter mitochondrial morphology during lytic replication

Epstein-Barr virus (EBV) is a human DNA virus that is responsible for the syndrome infectious mononucleosis, and is associated with several forms of cancer. During both lytic and latent viral infection, viral proteins manipulate the host's cellular components to aid in viral replication and maintenance. Here, it is demonstrated that induction of EBV lytic replication results in a dramatic reorganization of mitochondria accompanied by a significant alteration of mitochondrial membrane potential and a rapid and transient increase in the microtubular cytoskeleton. Moreover, we show that expression of the EBV immediate-early genes BZLF1 and BRLF1 contributes to the mitochondrial alteration but not the increase in the microtubule cytoskeleton, suggesting that the mechanism for the observed cytoplasmic restructuring involves a number of coordinated viral and host proteins.