分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 2016-05-12
Chen, Yongzhi
He, Liang
Peng, Yanan
Shi, Xiaodong
Cheng, Genhong
Deng, Hongyu
Chen, Yongzhi
He, Liang
Peng, Yanan
Chen, Jizheng
Chen, Xinwen
Zhong, Jin
Cheng, Genhong
摘要： The transcription factor nuclear factor kB (NF-kappa B) is crucial for innate immune defense against viral infections, and its activation requires the ubiquitylation of upstream proteins, including the adaptor protein NEMO (NF-kappa B essential modulator). Many infectious pathogens, including hepatitis C virus (HCV), inhibit NF-kappa B signaling in host cells, which promotes pathogen survival. Frequently, HCV-infected individuals develop a chronic infection, which suggests that HCV can subvert host antiviral responses. We found that HCV infection and replication inhibited the activation of NF-kappa B by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), which was mediated by the viral protein NS3 and, to a lesser extent, NS5B. NS3 directly interacted with linear ubiquitin chain assembly complex (LUBAC), competed with NEMO for binding to LUBAC, and inhibited the LUBAC-mediated linear ubiquitylation of NEMO and the subsequent activation of NF-kappa B. Together, our results highlight an immune evasion strategy adopted by HCV to modulate host antiviral responses and enhance virus survival and persistence.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
Qi, Jing
Han, Chuanhui
Gong, Danyang
Liu, Ping
Zhou, Sheng
Deng, Hongyu
Qi, Jing
Han, Chuanhui
Gong, Danyang
Zhou, Sheng
摘要： Replication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit of ORF48 from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates the ORF48 promoter via an RTA-responsive element (48pRRE). RTA binds to 48pRRE directly in vitro and also associates with ORF48 promoter in vivo. Mutagenesis of 48pRRE in the context of the viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE during de novo infection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48 in vitro and in vivo. The ORF48-null mutation remarkably reduced the viral replication efficiency in cell culture. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung and establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identified ORF48 as an RTA-responsive gene and showed that ORF48 is important for MHV-68 replication both in vitro and in vivo. IMPORTANCE The replication and transcription activator (RTA), conserved among gammaherpesviruses, serves as a molecular switch for the virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identified ORF48 as an RTA-responsive gene of MHV-68 and mapped the cis element involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of viral infection in vivo. Our study provides insights into the transcriptional regulation and protein function of MHV-68, a desired model for studying gammaherpesviruses.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
摘要： Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in the nuclear egress of capsids into the cytoplasm, especially for the two human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to a lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study the de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remains unclear how ORF33 is incorporated into virions. In this study, we first show that the endogenous ORF33 protein colocalizes with capsid proteins at discrete areas in the nucleus during viral infection. Cosedimentation analysis as well as an immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. An immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we show that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 to be a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner. IMPORTANCE Morphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although the process of capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between the capsid and the envelope in a herpesvirus virion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions in both the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we show that ORF33 is associated with intranuclear capsids prior to primary envelopment and identify novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids at an early stage of the virion maturation process for herpesviruses.
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