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Background: Duck enteritis virus (DEV) belongs to Alphaherpesvirinae; little is known about the DEV UL7 gene and its encoded protein. This study examined the molecular characteristics of DEV pUL7 in vitro and determined whether DEV pUL7 co-localizes with pUL51. Results: The results showed that UL7 can be regarded as a late gene. Moreover, immunofluorescence assay revealed that pUL7 was located around the perinuclear cytoplasmic region and co-localized with pUL51 in the cytoplasm and nucleus after transfection into duck embryo fibroblast cells (DEFs). Conclusion: In conclusion, we identified the molecular characteristics of the DEV UL7 gene, which is a late gene, and the co-localization of its encoded protein with pUL51 in transfected DEFs, enriching our understanding of pUL7 and future research directions. [ABSTRACT FROM AUTHOR]

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi's sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. The release of infectious virions requires the production of capsid proteins and other late genes, whose production is transcriptionally controlled by a complex of six virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.

ABSTRACT: Duck plague virus (DPV) pUL48 is a homologous of herpes simplex virus VP16, and some studies have shown that VP16 is essential for viral replication and proliferation, but there are few studies on DPV pUL48. Therefore, in order to study the function of pUL48 protein, we constructed a UL48-deleted mutant (DPV-BAC-∆UL48) that completely reemoved the UL48 gene from the DPV BAC genome and the revertant virus (DPV-BAC-∆UL48R) by using the 2-step red recombination system. Compared with the parental virus (DPV-BAC) and the revertant virus, the titer of UL48-deleted mutant was reduced by more than 38.2%, and the efficiency of producing infectious virions was significantly reduced. In addition, the average size of plaques produced by UL48-deleted mutant was about 30% smaller than that of the parental and revertant viruses, suggesting that pUL48 protein affected the cell-to-cell transmission of DPV. Finally, pharmacological inhibition assay showed that pUL48 is a late protein of DPV. In this study, we found that UL48, as a late gene, plays an important role in viral replication by affecting the formation of DPV infectious virion, virus cell-to-cell transmission, and viral genome transcription, which may provide some help for the study of the function of DPV pUL48 protein and the prevention and control of DPV.

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

Abstract Background pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Results We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.

Late gene expression of betaherpesviruses and gammaherpesviruses is tightly controlled by virus-encoded transactivation factors (vTFs). We recently proved that the 6 vTFs of murine cytomegalovirus (MCMV) form a complex to regulate late gene transcription. pM49, one of the vTFs that has not been studied before, was identified to be a component of the complex that interacts with pM95. In this study, we began to investigate the potential role of pM49 in viral late gene expression. A recombinant MCMV expressing C-terminal FLAG-tagged pM49 was constructed to study the expression kinetics and localization of pM49. pM49 was expressed at the late time of virus infection. Inhibition of viral DNA synthesis by phosphonate sodium phosphonic acid (PAA) abolished pM49 expression, indicating that it is a late protein. pM49 colocalized with pM44 at the viral replication compartment, similarly to other viral vTFs that have been reported. Mutant virus lacking full-length pM49 expression failed to express viral late genes, leading to nonproductive infection. The expression of immediate early and early genes was not affected, and viral DNA synthesis was only minimally affected during pM49-deficient virus infection. All of these data support the role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression. IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases. [ABSTRACT FROM AUTHOR]

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi's sarcomaassociated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteinerich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters. [ABSTRACT FROM AUTHOR]

Background: pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Results: We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions: The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21. [ABSTRACT FROM AUTHOR]

Vaccinia virus (VACV), the prototypical member of the poxvirus family, was used as a live-virus vaccine to eradicate smallpox worldwide and has recently received considerable attention because of its potential as a prominent vector for the development of vaccines against infectious diseases and as an oncolytic virus for cancer therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigen-presenting cells (APCs), including monocytes, macrophages, and dendritic cells. However, very few studies have assessed the interactions of VACV with primary human B cells, a main type of professional APCs. In this study, we evaluated the susceptibility of primary human peripheral B cells at various differentiation and maturation stages to VACV binding, infection, and replication. We found that plasmablasts were resistant to VACV binding, while other B subsets, including transitional, mature naive, memory, and plasma cells, were highly susceptible to VACV binding. VACV binding preference was likely associated with differential expression of chemokine receptors, particularly CXCR5. Infection studies showed that plasmablast, plasma, transitional, and mature naive B cells were resistant to VACV infection, while memory B cells were preferentially infected. VACV infection in ex vivo B cells was abortive, which occurred at the stage of late viral gene expression. In contrast, activated B cells were permissive to productive VACV infection. Thus, primary human B cells at different differentiation stages exhibit distinct susceptibilities to VACV binding and infection, and the infections are abortive and productive in ex vivo and activated B cells, respectively. [ABSTRACT FROM AUTHOR]

Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in betaand gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression. IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies. [ABSTRACT FROM AUTHOR]

Duck plague virus (DPV) pUL48 is a homologous of herpes simplex virus VP16, and some studies have shown that VP16 is essential for viral replication and proliferation, but there are few studies on DPV pUL48. Therefore, in order to study the function of pUL48 protein, we constructed a UL48-deleted mutant (DPV-BAC-∆UL48) that completely reemoved the UL48 gene from the DPV BAC genome and the revertant virus (DPV-BAC-∆UL48R) by using the 2-step red recombination system. Compared with the parental virus (DPV-BAC) and the revertant virus, the titer of UL48-deleted mutant was reduced by more than 38.2%, and the efficiency of producing infectious virions was significantly reduced. In addition, the average size of plaques produced by UL48-deleted mutant was about 30% smaller than that of the parental and revertant viruses, suggesting that pUL48 protein affected the cell-to-cell transmission of DPV. Finally, pharmacological inhibition assay showed that pUL48 is a late protein of DPV. In this study, we found that UL48 , as a late gene, plays an important role in viral replication by affecting the formation of DPV infectious virion, virus cell-to-cell transmission, and viral genome transcription, which may provide some help for the study of the function of DPV pUL48 protein and the prevention and control of DPV. [ABSTRACT FROM AUTHOR]

During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that can be divided into three general stages: immediate-early (IE), early (E) and late (L). This expression program is the result of a complex interplay between viral and cellular factors at both the transcriptional and post-transcriptional levels, as well as structural differences within the promoter architecture for each of the three gene classes. Since the cellular enzyme RNA polymerase II (RNAP-II) is responsible for the transcription of herpesvirus genes, most viral promoters contain DNA motifs that are common with those of cellular genes, although promoter complexity decreases from immediate-early to late genes. Immediate-early and early promoters contain numerous cellular and viral cis-regulating sequences upstream of a TATA box, whereas late promoters differ significantly in that they lack cis-acting sequences upstream of the Transcription Start Site (TSS). Moreover, in the case of the β- and γ-herpesviruses, a TATT box motif is frequently found in the position where the consensus TATA box of eukaryotic promoters usually localizes. The mechanisms of transcriptional regulation of the late viral gene promoters appear to be different between α-herpesviruses and the two other herpesvirus subfamilies ( and ). In this review, we will compare the mechanisms of late gene transcriptional regulation between HSV-1, for which the viral IE transcription factors - especially ICP4 - play an essential role, and the two other subfamilies of herpesviruses, with a particular emphasis on EBV, which has recently been found to code for its own specific TATT-binding protein.

During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that can be divided into three general stages: immediate-early (IE), early (E), and late (L). This expression program is the result of a complex interplay between viral and cellular factors at both the transcriptional and post-transcriptional levels, as well as structural differences within the promoter architecture for each of the three gene classes. Since the cellular enzyme RNA polymerase II (RNAP-II) is responsible for the transcription of herpesvirus genes, most viral promoters contain DNA motifs that are common with those of cellular genes, although promoter complexity decreases from immediate-early to late genes. Immediate-early and early promoters contain numerous cellular and viral cis-regulating sequences upstream of a TATA box, whereas late promoters differ significantly in that they lack cis-acting sequences upstream of the transcription start site (TSS). Moreover, in the case of the β- and γ-herpesviruses, a TATT box motif is frequently found in the position where the consensus TATA box of eukaryotic promoters usually localizes. The mechanisms of transcriptional regulation of the late viral gene promoters appear to be different between α-herpesviruses and the two other herpesvirus subfamilies (β and γ). In this review, we will compare the mechanisms of late gene transcriptional regulation between HSV-1, for which the viral IE transcription factors - especially ICP4 - play an essential role, and the two other subfamilies of herpesviruses, with a particular emphasis on EBV, which has recently been found to code for its own specific TATT-binding protein. [ABSTRACT FROM AUTHOR]

Late gene expression of betaherpesviruses and gammaherpesviruses is tightly controlled by virus-encoded transactivation factors (vTFs). We recently proved that the 6 vTFs of murine cytomegalovirus (MCMV) form a complex to regulate late gene transcription. pM49, one of the vTFs that has not been studied before, was identified to be a component of the complex that interacts with pM95. In this study, we began to investigate the potential role of pM49 in viral late gene expression. A recombinant MCMV expressing C-terminal FLAG-tagged pM49 was constructed to study the expression kinetics and localization of pM49. pM49 was expressed at the late time of virus infection. Inhibition of viral DNA synthesis by phosphonate sodium phosphonic acid (PAA) abolished pM49 expression, indicating that it is a late protein. pM49 colocalized with pM44 at the viral replication compartment, similarly to other viral vTFs that have been reported. Mutant virus lacking full-length pM49 expression failed to express viral late genes, leading to nonproductive infection. The expression of immediate early and early genes was not affected, and viral DNA synthesis was only minimally affected during pM49-deficient virus infection. All of these data support the role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression. IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases., (Copyright © 2020 American Society for Microbiology.)

Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression. IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies., (Copyright © 2018 American Society for Microbiology.)