- Volume 91, Issue 9, 2010

Multiple proteins interacting with DNA polymerases orchestrate DNA replication. Human cytomegalovirus (HCMV) encodes a DNA polymerase that includes the presumptive processivity factor UL44. UL44 is structurally homologous to the eukaryotic DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous proteins. Previous proteomic analysis has identified the HCMV protein IRS1 as a candidate protein interacting with UL44. Nuclease-resistant reciprocal co-immunoprecipitation of UL44 with IRS1 and with TRS1, which has an amino terminus identical to that of IRS1, was observed from lysate of cells infected with viruses expressing epitope-tagged UL44, epitope-tagged IRS1 or epitope-tagged TRS1. Western blotting of protein immunoprecipitated from infected cell lysate indicated that epitope-tagged IRS1 and TRS1 do not associate simultaneously with UL44. Glutathione S-transferase pull-down experiments indicated that IRS1 and TRS1 interact with UL44 via a region that is identical in both proteins. Taken together, these data suggest that IRS1 and TRS1 may compete for association with UL44 and may affect UL44 function differentially.

Cancers with viral aetiologies can potentially be prevented by antiviral vaccines. Therefore, it is important to understand how viral infections and cancers might be linked. Some cancers frequently carry gammaherpesvirus genomes. However, they generally express the same viral genes as non-transformed cells, and differ mainly in also carrying oncogenic host mutations. Infection, therefore, seems to play a triggering or accessory role in disease. The hit-and-run hypothesis proposes that cumulative host mutations can allow viral genomes to be lost entirely, such that cancers remaining virus-positive represent only a fraction of those to which infection contributes. This would have considerable implications for disease control. However, the hit-and-run hypothesis has so far lacked experimental support. Here, we tested it by using Cre–lox recombination to trigger transforming mutations in virus-infected cells. Thus, ‘floxed’ oncogene mice were infected with Cre recombinase-positive murid herpesvirus-4 (MuHV-4). The emerging cancers showed the expected genetic changes but, by the time of presentation, almost all lacked viral genomes. Vaccination with a non-persistent MuHV-4 mutant nonetheless conferred complete protection. Equivalent human gammaherpesvirus vaccines could therefore potentially prevent not only viral genome-positive cancers, but possibly also some cancers less suspected of a viral origin because of viral genome loss.

The BGLF4 protein of Epstein–Barr virus (EBV) is a serine/threonine protein kinase that phosphorylates several viral and cellular substrates at cellular cyclin-dependent kinase target sites. BGLF4 is required for efficient viral DNA replication and release of mature virions. It also stimulates the transactivation activity of the immediate-early transactivator Zta (BZLF1) and suppresses the transactivation activities of BMRF1 and EBNA-2. This study aimed to characterize further the regulation of BGLF4 expression at the transcriptional and translational levels. It was shown that BGLF4 was expressed with early kinetics and reached maximal levels after DNA replication. The promoter activity of BGLF4 was upregulated mainly by the immediate-early transactivator Rta, rather than Zta, as revealed by Zta-specific short hairpin RNA in EBV-positive cells and by luciferase reporter assays. By rapid amplification of 5′ cDNA ends, two major transcriptional start sites were identified at 201 and 255 nt upstream of the first in-frame ATG of BGLF4 in P3HR1 cells. An additional transcript initiated from −468 was detected in Akata cells. The translation initiation site of BGLF4 was confirmed by mutagenesis, in vitro translation and transient transfection. The translation regulatory effect mediated by the long 5′-untranslated region (5′UTR) of BGLF4 was demonstrated by dual reporter assays in 293T and EBV-positive NA cells. These results suggested that different promoter usage and 5′UTR-mediated translation enhancement may ensure the proper expression of BGLF4 at various stages of virus replication.

In cell lines, the Epstein–Barr virus (EBV)-encoded protein latent membrane protein 2A (LMP2A) protects B-cells from apoptosis by blocking B-cell receptor (BCR) signalling. However, EBV-infected B-cells in vivo are extremely different from cell lines. This study used a murine transgenic model in which B-cells express LMP2A and a BCR specific for hen egg lysozyme to determine whether LMP2A protects resting and antigen-activated B-cells from apoptosis. LMP2A allows BCR signal transduction and induces constitutive activation of NF-κB to increase Bcl-2 levels that afford LMP2A-mediated protection from apoptosis in the absence or presence of antigen. In contrast, low levels of NF-κB inhibitor only affected Bcl-2 and Bcl-xL levels and increased apoptosis in LMP2A-negative B-cells after BCR cross-linking. These data suggest that LMP2A uniquely makes resting B-cells sensitive to NF-κB inhibition and apoptosis and suggest that NF-κB may be a novel target to eradicate latently EBV-infected B-cells.

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus functions as an origin-binding protein (OBP) and transcriptional regulator. LANA binds the terminal repeats via the C-terminal DNA-binding domain (DBD) to support latent DNA replication. To date, the structure of LANA has not been solved. Sequence alignments among OBPs of gammaherpesviruses have revealed that the C terminus of LANA is structurally related to EBNA1, the OBP of Epstein–Barr virus. Based on secondary structure predictions for LANADBD and published structures of EBNA1DBD, this study used bioinformatics tools to model a putative structure for LANADBD bound to DNA. To validate the predicted model, 38 mutants targeting the most conserved motifs, namely three α-helices and a conserved proline loop, were constructed and functionally tested. In agreement with data for EBNA1, residues in helices 1 and 2 mainly contributed to sequence-specific DNA binding and replication activity, whilst mutations in helix 3 affected replication activity and multimer formation. Additionally, several mutants were isolated with discordant phenotypes, which may aid further studies into LANA function. In summary, these data suggest that the secondary and tertiary structures of LANA and EBNA1 DBDs are conserved and are critical for (i) sequence-specific DNA binding, (ii) multimer formation, (iii) LANA-dependent transcriptional repression, and (iv) DNA replication.

Vaccinia virus (VACV) encodes multiple proteins to evade host innate immunity, including B14, a virulence factor that binds to the inhibitor of κB kinase β (IKKβ) and blocks nuclear factor κB (NF-κB) activation. B14 shares 95 % amino acid identity with the 183 protein encoded by modified virus Ankara (MVA), an attenuated VACV strain being developed as a vaccine vector. To evaluate whether the immunogenicity of MVA might be increased by manipulation of MVA immunomodulatory proteins, the MVA counterpart of B14, protein 183, was characterized. Unlike B14, protein 183 was unstable in eukaryotic cells unless proteasome-mediated protein degradation was inhibited. Furthermore, 183 did not inhibit NF-κB activation in response to cytokine stimulation, and did not restore the virulence of VACV strain Western Reserve lacking gene B14R. The instability and non-functionality of 183 are probably explained by a deletion of 6 aa within α-helix 6 of the B14 crystal structure.

The vaccinia virus E3 protein is an important intracellular modulator of innate immunity that can be split into distinct halves. The C terminus contains a well defined dsRNA-binding domain, whereas the N terminus contains a Z-DNA-binding domain, and both domains are required for virulence. In this study, we investigated whether the E3 Z-DNA-binding domain functions by sequestering cytoplasmic dsDNA thereby preventing the induction of type I interferon (IFN). In line with this hypothesis, expression of E3 ablated both IFN-β expression and NF-κB activity in response to the dsDNA, poly(dA–dT). However, surprisingly, the ability of E3 to block poly(dA–dT) signalling was independent of the N terminus, whereas the dsRNA-binding domain was essential, suggesting that the Z-DNA-binding domain does not bind immunostimulatory dsDNA. This was confirmed by the failure of E3 to co-precipitate with biotinylated dsDNA, whereas the recruitment of several cytoplasmic DNA-binding proteins could be detected. Recently, AT-rich dsDNA was reported to be transcribed into 5′-triphosphate poly(A-U) RNA by RNA polymerase III, which then activates retinoic acid-inducible gene I (RIG-I). Consistent with this, RNA from poly(dA–dT) transfected cells induced IFN-β and expression of the E3 dsRNA-binding domain was sufficient to ablate this response. Given the well documented function of the E3 dsRNA-binding domain we propose that E3 blocks signalling in response to poly(dA–dT) by binding to transcribed poly(A-U) RNA preventing RIG-I activation. This report describes a DNA virus-encoded inhibitor of the RNA polymerase III-dsDNA-sensing pathway and extends our knowledge of E3 as a modulator of innate immunity.