Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that inter- actions among tegument proteins play a key role in virion mor...Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that inter- actions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gam- maherpesviruses including Kaposi＇s sarcoma-associ- ated herpesvirus （KSHV） and Epstein-Barr virus （EBV） is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvi- rus-68 （MHV-68） is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion mor- phogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network （TGN）, a site where secondary envelopment takes place.展开更多
Murine gammaherpesvirus 68(MHV-68),a member of the gammaherpesvirus family,replicates robustly in permissive cell lines and is able to infect laboratory mice.MHV-68 has emerged as a model for studying the basic aspect...Murine gammaherpesvirus 68(MHV-68),a member of the gammaherpesvirus family,replicates robustly in permissive cell lines and is able to infect laboratory mice.MHV-68 has emerged as a model for studying the basic aspects of viral replication and host–virus interactions of its human counterparts.Herpesvirus genome replication is mediated through a cis-element in the viral genome called the origin of lytic replication(oriLyt).A family of transcription factors,CCAAT/enhancer binding proteins(C/EBPs),assists in oriLyt-mediated DNA replication during gammaherpesvirus reactivation.In this study,we examined the role of C/EBPs in gammaherpesvirus DNA replication during de novo infection,using MHV-68 as a model.We found that C/EBP α and β bind to the CCAAT boxes in the MHV-68 oriLyt core region both in vitro and in vivo,as demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay.A dominant negative form of C/EBPs significantly impaired the lytic replication efficiency of MHV-68 on both the plasmid and genome levels in a replication assay,indicating that functional C/EBPs are required for maximal MHV-68 genome DNA replication.Collectively,our data demonstrate that C/EBPs interact with the oriLyt core region and play an important role in MHV-68 lytic DNA replication during de novo infection.展开更多
基金We thank Drs. Ren Sun and Tao Xu for kindly providing plasmids, Mr. Shufeng Sun at the Center for Biological Imaging, Institute of Biophysics, for help with EM sample preparation and members of the Deng laboratory for helpful discussions. This work was sup- ported by grants from the National Natural Science Foundation of China （Grant No. 81171582） and the National Basic Research Program （973 Program） （No. 2011CB504300）.
文摘Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that inter- actions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gam- maherpesviruses including Kaposi＇s sarcoma-associ- ated herpesvirus （KSHV） and Epstein-Barr virus （EBV） is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvi- rus-68 （MHV-68） is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion mor- phogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network （TGN）, a site where secondary envelopment takes place.
基金supported by the National Natural Science Foundation of China(Grant No.30930007).
文摘Murine gammaherpesvirus 68(MHV-68),a member of the gammaherpesvirus family,replicates robustly in permissive cell lines and is able to infect laboratory mice.MHV-68 has emerged as a model for studying the basic aspects of viral replication and host–virus interactions of its human counterparts.Herpesvirus genome replication is mediated through a cis-element in the viral genome called the origin of lytic replication(oriLyt).A family of transcription factors,CCAAT/enhancer binding proteins(C/EBPs),assists in oriLyt-mediated DNA replication during gammaherpesvirus reactivation.In this study,we examined the role of C/EBPs in gammaherpesvirus DNA replication during de novo infection,using MHV-68 as a model.We found that C/EBP α and β bind to the CCAAT boxes in the MHV-68 oriLyt core region both in vitro and in vivo,as demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay.A dominant negative form of C/EBPs significantly impaired the lytic replication efficiency of MHV-68 on both the plasmid and genome levels in a replication assay,indicating that functional C/EBPs are required for maximal MHV-68 genome DNA replication.Collectively,our data demonstrate that C/EBPs interact with the oriLyt core region and play an important role in MHV-68 lytic DNA replication during de novo infection.
