Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

Only the HLA class I gene minimal promoter elements are required for transactivation by human cytomegalovirus immediate early genes

The immediate early (IE) genes of human cytomegalovirus (HCMV) are expressed in lymphocytes and are known to transactivate both viral and cellular promoters. The mechanism by which IE gene products of HCMV transactivate expression of the HLA A2 gene promoter in Jurkat cells, a T-lymphocyte cell line, was investigated. Transient expression assays were performed using plasmids containing the HLA A2 promoter-regulatory region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE genes. The upregulation of the HLA A2 promoter by HCVM IE gene products was shown not to be secondary to either interferon-gamma or -alpha. Previously described MHC class I regulatory or enhancer elements such as the interferon-stimulated response element (ISRE), NF-kappa B and H2TF1 binding sequences, and the interferon consensus sequence (ICS) were not required for transactivation of the A2 promoter. Rather, the only known regulatory elements in the HLA A2 promoter necessary for both basal expression and transactivation by HCVM IE gene products are the CCAAT box and TATA box motifs. These results support a model in which HCVM IE gene products act through the minimal HLA A2 promoter elements to increase gene expression.     

Both Epstein-Barr viral nuclear antigen 2 (EBNA2) and activated Notch1 transactivate genes by interacting with the cellular protein RBP-J kappa

The Epstein-Barr viral nuclear antigen 2 (EBNA2) plays a key role during establishment and maintenance of B cell immortalization after Epstein-Barr virus (EBV) infection. EBNA2 acts as a transactivator of cellular and viral genes. We studied two EBNA2 regulated viral promoters (TP1 promoter and LMP/TP2 promoter) in detail to learn more about the molecular mechanisms of EBNA2-mediated transactivation. In both promoters we could identify at least one binding site for the cellular repressor protein RBP-J kappa. EBNA2 is tethered to the EBNA2 responsive promoter elements by interaction with this cellular protein. Although necessary, the binding of RBP-J kappa is not sufficient for EBNA2-mediated transactivation. At least two further cellular proteins, which are different in the studied promoters are important for efficient transactivation. The identification of RBP-J kappa as central mediator of EBNA2 transactivation suggested an interference of EBNA2 with the highly conserved Notch receptor signal transduction pathway. We could show that an activated form of the Notch receptor can transactivate a reporter construct containing a hexamer of the two RBP-J kappa binding sites of the TP1 promoter supporting the idea that EBNA2 acts as a functional equivalent of an activated Notch receptor.     

NF-kappaB-mediated inhibition of apoptosis is required for encephalomyocarditis virus virulence: a mechanism of resistance in p50 knockout mice

Apoptosis is a central host defense mechanism to eliminate virus-infected cells. Activation of NF-kappaB suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine encephalomyocarditis virus (EMCV) infection in mice and cell lines defective in NF-kappaB1 (p50) signaling. As previously reported, we find that all p50 knockout (p50 -/-) mice survive an EMCV infection that readily kills normal mice. By introducing the p50 mutation into interferon (IFN) type I receptor knockout (IFNRI -/-) mice, we find that this resistance is not mediated by IFN-beta as previously thought. While no IFNRI -/- mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals' ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 -/-, and p65 -/- embryos, we found that NF-kappaB is not required for the replication cycle of EMCV. However, during these experiments we observed that p50 -/- and p65 -/- MEF infected with EMCV undergo enhanced, premature cytotoxicity. Upon examination of this cell death, we found that EMCV infection induced both plasma membrane and nuclear changes typical of apoptosis in all cell lines. These apoptotic processes occurred in an accelerated and pronounced way in the NF-kappaB-defective cells, as soon as 6 h after infection, when virus is beginning to be released. Previously, only the RelA (p65) subunit of NF-kappaB has been shown to play a role in suppressing apoptosis. In our studies, we find that p50 is equally important in suppressing apoptosis during EMCV infection. Additionally, we show that suppression of apoptosis by NF-kappaB1 is required for EMCV virulence in vivo. The attenuation in p50 -/- mice can be explained by rapid apoptosis of infected cells which allows host phagocytes to clear infected cells before the viral burst leading to a reduction of the viral burden and survival of the mice.     

Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein

Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.