Laurence Baudoux

Phosphorylation of Varicella-Zoster Virus IE63 Protein by Casein Kinases Influences Its Cellular Localization and Gene Regulation Activity

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivophosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or post-transcriptional level.

Intracellular Transport of the Glycoproteins gE and gI of the Varicella-Zoster Virus gE ACCELERATES THE MATURATION OF gI AND DETERMINES ITS ACCUMULATION IN THE TRANS-GOLGI NETWORK

The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses.

Intracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses. The major regulatory immediate-early protein of VZV (IE62) is a transactivator of VZV gene expression. In transfection assays, IE4 has been shown to enhance activation induced by IE62. To investigate the functional interactions underlying this observation, indirect immunofluorescence studies were undertaken to determine whether IE62 could influence IE4 intracellular localization in transfected cells. In single transfections, IE4 was predominantly found in cytoplasm. In cotransfection with IE62, the IE4 localization pattern was altered, with nuclear staining predominating over cytoplasmic staining. This effect was specific to the IE62 protein since the gene products of ORF63 and ORF61, which are also regulatory proteins, did not influence IE4 distribution. The use of IE62 mutants indicated that IE62 influence is independent of its transactivation function and that the integrity of regions 3 and 4 is required. IE62 remained nuclear whether IE4 was present or not. These observations underline differences in the regulation of gene expression between VZV proteins and their herpes simplex virus type 1 homologues. In infected cells, IE4 was only sometimes found to colocalize with IE62 in nuclei. This observation suggests that when all VZV proteins are present, complex interactions probably occur which could diminish the influence of IE62.

Gene activation by Varicella-zoster virus IE4 protein requires its dimerization and involves both the arginine-rich sequence, the central part, and the carboxyl-terminal cysteine-rich region.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors act as dimers, IE4 dimerization was studied using the mammalian two-hybrid system. Introduction of mutations in the IE4 open reading frame demonstrated that both the central region and the carboxyl-terminal cysteine-rich domain were important for efficient dimerization. Within the carboxyl-terminal domain, substitution of amino acids encompassing residues 443–447 totally abolished dimerization. Gene activation by IE4 was studied by transient transfection with an IE4 expression plasmid and a reporter gene under the control of either the human immunodeficiency virus, type 1, long terminal repeat or the VZV thymidine kinase promoter. Regions of IE4 important for dimerization were also shown to be crucial for transactivation. In addition, the arginine-rich domains Rb and Rc of the amino-terminal region were also demonstrated to be important for transactivation, whereas the Ra domain as well as an acidic and bZIP-containing regions were shown to be dispensable for gene transactivation. A nucleocytoplasmic shuttling of IE4 has also been characterized, involving a nuclear localization signal identified within the Rb domain and a nuclear export mechanism partially depending on Crm-1.

Lessons to be learned from varicella-zoster virus

Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the skin where it causes typical lesions. There, it penetrates the peripheral nervous system and it remains latent in dorsal root ganglia. It is still debatable whether VZV persists in neurons or in satellite cells. During latency, VZV expresses a limited set of transcripts of its immediate early (IE) and early (E) genes but no protein has been detected. Mechanisms of reactivation from ganglia have not been identified. However, dysfunction of the cellular immune system appears to be involved in this process. The cell-associated nature of VZV has made it difficult to identify a temporal order of gene expression, but there appears to be a cascade mechanism as for HSV-1. The lack of high titre cell-free virions or recombination mutants has hindered so far the understanding of VZV gene functions. Five genes, ORFs 4, 10, 61, 62, and 63 that encode regulatory proteins could be involved in VZV latency. ORF4p activates gene promoters with basal activities. ORF10p seems to activate the ORF 62 promoter. ORF61p has trans-activating and trans-repressing activities. The major IE protein ORF62p, a virion component, has DNA-binding and regulatory functions, transactivates many VZV promoters and even regulates its own expression. ORF63p is a nuclear IE protein of yet unclear regulatory functions, abundantly expressed very early in infection. We have established an animal model of VZV latency in the rat nervous system, enabling us to study the expression of viral mRNA and protein expression during latency, and yielding results similar to those found in humans. This model is beginning to shed light on the molecular events in VZV persistent infection and on the regulatory mechanisms that maintain the virus in a latent stage in nerve cells.

Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins

Varicella‐Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate‐early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate‐early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate‐early promoter. VZV OKA infection of these stably transformed cell lines was followed‐up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5‐ to 10‐fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2‐ to 5‐fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle.

Varicella-zoster virus gene regulation

The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein of the virus exhibiting DNA-binding and regulatory functions. This protein, localized in the cell nucleus, is a functional homologue to ICP4 of herpes simplex virus type 1 (HSV-1). It trans-activates several varicella-zoster virus promoters of the various gene classes and autoregulates its own expression. ORF4 protein activates gene promoters provided they have basal activities, but it is not a functional homologue of HSV-1 ICP27. Gene regulation activity appears to be linked to its cysteine-rich C-terminal region. ORF63 codes for an immediate early protein mainly located in the cell nucleus. The regulatory functions it performs are still unclear. ORF61 protein is the functional homologue of HSV-1 ICP0. Its N-terminal region exhibits a RING domain responsible for trans-activating and trans-repressing activities. ORF10 protein exhibits similarities with HSV-1 VP16 and activates the ORF62 promoter. NEUROLOGY 1995;45(Suppl 8): S23-S27