DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.     

Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein – Barr virus early promoter, BMRF1

Disruption of Epstein – Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243 – 9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085 – 4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at − 59 and − 106), whereas two additional Z binding sites (located at − 42 and − 170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251 – 8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.     

Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1

The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.     

Identifying the key genes of Epstein–Barr virus‐regulated tumour immune microenvironment of gastric carcinomas

The Epstein–Barr virus (EBV) is involved in the carcinogenesis of gastric cancer (GC) upon infection of normal cell and induces a highly variable composition of the tumour microenvironment (TME). However, systematic bioinformatics analysis of key genes associated with EBV regulation of immune infiltration is still lacking. In the present study, the TCGA and GEO databases were recruited to analyse the association between EBV infection and the profile of immune infiltration in GC. The weighted gene co‐expression analysis (WGCNA) was applied to shed light on the key gene modules associated with EBV‐associated immune infiltration in GC. 204 GC tissues were used to analysed the expression of key hub genes by using the immunohistochemical method. Real‐time PCR was used to evaluate the association between the expression of EBV latent/lytic genes and key immune infiltration genes. Our results suggested that EBV infection changed the TME of GC mainly regulates the TIICs. The top three hub genes of blue (GBP1, IRF1, and LAP3) and brown (BIN2, ITGAL, and LILRB1) modules as representative genes were associated with EBV infection and GC immune infiltration. Furthermore, EBV‐encoded LMP1 expression is account for the overexpression of GBP1 and IRF1. EBV infection significantly changes the TME of GC, and the activation of key immune genes was more dependent on the invasiveness of the whole EBV virion instead of single EBV latent/lytic gene expression.

The lytic EBV virion infects host cells, activates the immune response of the host and promotes the expression of associated key hub genes. Then EBV establishes latent infection in host cells and expressed LMP1, which promotes activation of the NF‐κB signalling pathway and expression of GBP1 and IRF1.     

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein‐Barr virus orthologs

Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein‐Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX‐TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine‐rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co‐ordinate nuclear and cytoplasmic processes.      

Structural basis of the association of HIV-1 matrix protein with DNA

HIV-1 matrix (MA) is a multifunctional protein that is synthesized as a polyprotein that is cleaved by protease during viral maturation. MA contains a cluster of basic residues whose role is controversial. Proposed functions include membrane anchoring, facilitating viral assembly, and directing nuclear import of the viral DNA. Since MA has been reported to be a component of the preintegration complex (PIC), we have used NMR to probe its interaction with other PIC components. We show that MA interacts with DNA and this is likely sufficient to account for its association with the PIC.