Direct stimulation of translation by the multifunctional herpesvirus ICP27 protein.

Herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential regulator of viral gene expression with roles at various levels of RNA metabolism in the nucleus. Using the tethered function assay, we showed a cytoplasmic activity for ICP27 in directly enhancing mRNA translation in vivo in the absence of other viral factors. The region of ICP27 required for translational stimulation maps to the C terminus. Furthermore, in infected cells, ICP27 is associated with polyribosomes, indicating a function in translation during the lytic cycle.

Protein kinase CK2 phosphorylation regulates the interaction of Kaposi's sarcoma-associated herpesvirus regulatory protein ORF57 with its multifunctional partner hnRNP K.

ORF57 protein of Kaposi's sarcoma-associated herpesvirus has a counterpart in all herpesvirus of mammals and birds and regulates gene expression at transcriptional and post-transcriptional levels. ORF57 was capable of self-interaction and bound a rapidly migrating form of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional cellular protein involved in gene expression. In virus infected cell extracts, ORF57 was present in a complex with hnRNP K that had protein kinase CK2 activity, and was phosphorylated by CK2. Different regions of ORF57 bound both catalytic alpha/alpha' and regulatory beta subunits of CK2. CK2 modification enhanced the ORF57-hnRNP K interaction, and may regulate the presence and activities of components in the complex. We suggest that ORF57 and hnRNP K interaction may modulate ORF57-mediated regulation of viral gene expression. Herpesviral ORF57 (Rhadinovirus) and ICP27 (Simplexvirus) proteins both interact with hnRNP K and CK2 implying that adaptation of the ancestral hnRNP K and CK2 to associate with viral regulatory ancestor protein likely pre-dates divergence of these Herpesviridae genera that occurred 200 million years ago.

Functional co-operation between the Kaposi's sarcoma-associated herpesvirus ORF57 and ORF50 regulatory proteins.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) proteins ORF57 (also known as MTA) and ORF50 (also known as RTA) act post-transcriptionally and transcriptionally to regulate viral lytic gene expression and synergistically activate certain early and late KSHV promoters. When ORF57 and ORF50 were co-expressed, they co-operatively stimulated expression from the promoter of the immediate-early ORF50 gene itself. Co-immunoprecipitations with extracts of KSHV-infected cells showed that ORF57 and ORF50 proteins were present in the same complex. Using the pull-down assay with extracts of KSHV-infected cells, ORF50 protein was shown to interact with a glutathione S-transferase-ORF57 fusion protein. A chromatin immunoprecipitation assay showed that ORF50 promoter sequences were preferentially associated with immunoprecipitated chromatin using both anti-ORF50 and anti-ORF57 antibodies consistent with both an in vivo physical association between ORF57 and ORF50 and a potential role for ORF57 at the transcriptional level. This is the first demonstration of an interaction between these two lytic regulatory proteins in a gammaherpesvirus. Expression of ORF50 protein is sufficient to induce lytic replication in latently infected cells and may determine viral host range, spread and KS pathogenesis in vivo. A new insight into the co-ordinated activities of these two key regulatory proteins is provided in which upregulation of the ORF50 promoter with augmentation of ORF50 activity by ORF57 protein, and vice versa, would facilitate the cascade of lytic viral gene expression, thereby breaking latency. A functional and physical interaction between these two gammaherpesvirus regulatory protein counterparts could be a general feature of the herpesviruses.

The evolutionarily conserved Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with REF protein and acts as an RNA export factor.

ORF57 (MTA) one of the earliest Kaposi's sarcoma-associated herpesvirus (KSHV) regulatory proteins to be expressed is essential for virus lytic replication. A counterpart is present in every herpesvirus sequenced, indicating the importance of this signature viral protein and those examined act post-transcriptionally, affecting RNA splicing and transport. In KSHV-infected cells, ORF57 protein was present in a complex with REF (Aly) and TAP (NXF1), factors involved in cellular mRNA export. The ORF57 N-terminal region interacts with REF, whereas both N- and C-terminal domains of REF interact with ORF57. The ORF57-REF interaction was direct, whereas TAP appeared to be recruited via REF. In somatic cells, ectopically expressed ORF57 protein was shown to function as a CRM1-independent nuclear mRNA export factor, promoting export of mRNAs that are poor substrates for splicing. The gamma-herpesvirus ORF57 protein, and its alpha-1 herpesvirus ICP27 counterpart both export RNA through pathways involving REF and TAP proteins, although divergence of these herpesvirus subfamilies occurred some 180-210 million years ago. The TAP-mediated cellular mRNA export pathway is CRM1-independent. However, human immunodeficiency virus type 1 Rev protein-mediated RNA export, which is CRM1-dependent, was considerably inhibited by ORF57, suggesting that Rev and ORF57 compete for a common export component. These data strengthen arguments that TAP and CRM1 pathways converge in accessing similar components of the nuclear pore complex. We propose that ORF57-mediated RNA export may use different export factors to accommodate the KSHV-infected host cell environments, for example, in B-cells or endothelial cells and during the different phases of lytic virus replication.

Formation of nuclear foci of the herpes simplex virus type 1 regulatory protein ICP4 at early times of infection: localization, dynamics, recruitment of ICP27, and evidence for the de novo induction of ND10-like complexes.

Herpes simplex virus type 1 (HSV-1) has an intricate association with cellular nuclear structures known as ND10 or promyelocytic leukemia protein (PML) nuclear bodies. Parental viral genomes initially become juxtaposed to ND10, and then viral replication compartments develop from the ND10-associated genomes. Viral immediate-early (IE) regulatory protein ICP0 colocalizes with ND10 and then induces the degradation of critical ND10 component protein PML and therefore the release and dispersal of other ND10 proteins. The IE transcriptional regulatory protein ICP4 also forms foci at early times of infection, many of which are juxtaposed to ND10 and later develop into replication compartments, indicating that at least some of the initial ICP4 foci contain parental viral genomes. Here we report that the ICP4 foci also contain ICP27 and that their formation occurs extremely rapidly at locations just inside the nuclear envelope. By examining developing plaques or thinly seeded cells infected at high multiplicity, we found evidence to suggest that at least some of the ND10-viral nucleoprotein complex association could be attributed to de novo formation of ND10-like structures in response to incoming viral genomes. The ICP4 complexes associated efficiently with ND10 in cells infected with an ICP0-null mutant virus at high but not at low multiplicity, and the degree of association was reduced by the proteasome inhibitor MG132. Therefore, the interaction between viral nucleoprotein complexes and ND10 is in part due to a dynamic response by the cell. This response is modulated by functional ICP0, and cells that are productively or nonproductively infected in the absence of functional ICP0 can be distinguished by the relative locations of ICP4 foci and ND10 proteins.

Identification of herpes simplex virus RNAs that interact specifically with regulatory protein ICP27 in vivo.

Herpes simplex virus type 1 (HSV-1) protein ICP27 has an essential regulatory role during viral replication, in part by post-transcriptional control of gene expression, and has a counterpart in all herpes viruses sequenced so far. Although much is known about the functions of this signature herpesvirus protein, little is known about its RNA binding capabilities; ICP27 interacts with specificity for a subset of intronless HSV-1 RNAs and poly(G), through its RGG box. We performed an in vivo yeast three-hybrid screen of an HSV-1 genomic library, searching for ICP27 interacting RNAs. Comparable with a yeast genomic screen, 24 of 55 single inserts mapped to antisense strands of HSV-1 transcribed regions or non-transcribed regions. The 31 HSV-1 sense RNAs identified were 35 to 225 nucleotides in length and interacted with preferred specificity for ICP27 as compared with an unrelated RNA-binding protein. They map to 10 monocistronic and 10 polycistronic transcripts of all kinetic classes and represent 28 open reading frames encoding predominantly essential viral proteins with roles in viral DNA replication and virion maturation. Several studies show regulatory effects by ICP27 on the majority of these transcripts, consistent with its regulation of the early-late switch in the HSV-1 life cycle. Deletion of the ICP27 RGG box and the ICP27 M15 mutation, both lethal in virus, abolished or severely reduced the ICP27-RNA interactions, indicating their biological relevance. The study facilitates continued study of gene regulation by ICP27 by further defining its interactions with viral RNAs.

CK2 protein kinase is stimulated and redistributed by functional herpes simplex virus ICP27 protein.

It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs.

The herpes simplex virus type 1 US11 protein binds the coterminal UL12, UL13, and UL14 RNAs and regulates UL13 expression in vivo.

The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. US11 localizes to the nucleolus in infected cells, can associate with ribosomes, and has been shown to bind RNA. The RNA substrates of US11 identified thus far have no apparent role in the virus lytic cycle, so we set out to identify a novel, biologically relevant RNA substrate(s) for this protein in HSV-1-infected cells. We designed a reverse transcriptase PCR-based protocol that allowed specific selection of a 600-bp RNA binding partner for US11. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14. We show that the binding of US11 to 12/14 is sequence-specific and mediated by the C-terminal domain of the protein. To elucidate the role of US11 in the virus life cycle, we infected cells with wild-type virus, a cosmid-reconstructed US11 HSV-1 null mutant, and a cosmid-reconstructed wild-type virus and analyzed expression of UL12, -13, and -14 during a time course of infection. These experiments revealed that this interaction has biological activity; at early times of infection, US11 down-regulates UL13 protein kinase mRNA and protein.

The human papillomavirus type 31 late 3' untranslated region contains a complex bipartite negative regulatory element.

The papillomavirus life cycle is tightly linked to epithelial cell differentiation. Production of virus capsid proteins is restricted to the most terminally differentiated keratinocytes in the upper layers of the epithelium. However, mRNAs encoding the capsid proteins can be detected in less-differentiated cells, suggesting that late gene expression is controlled posttranscriptionally. Short sequence elements (less than 80 nucleotides in length) that inhibit gene expression in undifferentiated epithelial cells have been identified in the late 3' untranslated regions (UTRs) of several papillomaviruses, including the high-risk mucosal type human papillomavirus type 16 (HPV-16). Here we show that closely related high-risk mucosal type HPV-31 also contains elements that can act to repress gene expression in undifferentiated epithelial cells. However, the HPV-31 negative regulatory element is surprisingly complex, comprising a major inhibitory element of approximately 130 nucleotides upstream of the late polyadenylation site and a minor element of approximately 110 nucleotides mapping downstream. The first 60 nucleotides of the major element have 68% identity to the negative regulatory element of HPV-16, and these elements bind the same cellular proteins, CstF-64, U2AF(65), and HuR. The minor inhibitory element binds some cellular proteins in common with the major inhibitory element, though it also binds certain proteins that do not bind the upstream element.