The varicella-zoster virus (VZV) genes ORF47 and ORF66 are predicted to encode serine͞threonine protein kinases, which are homologs of herpes simplex virus 1 (HSV-1) UL13, and US3. When mutants were constructed by inserting stop codons into ORF47 and ORF66, the recombinants ROka47S and ROka66S, as well as intact ROka replicated in tissue culture. In contrast, inoculation of human thymus͞liver or skin implants in SCID-hu mice showed that ORF47 protein was required for viral growth in human T cells and skin. Eliminating ORF66 expression inhibited VZV infectivity for T cells partially but did not impair replication in skin compared with ROka. Infectivity for T cells and skin was restored when ROka47S virus was complemented by insertion of ORF47 into a distant, noncoding site. The ORF47 gene product is the first VZV protein identified as necessary for T cell tropism. It also is essential for skin infectivity in vivo, as is glycoprotein C. Expression of ORF66 did not compensate for the absence of the ORF47 protein. The requirement for ORF47 expression in T cells and skin indicates that this gene product, which is dispensable in vitro, has a critical role within differentiated cells that are essential targets for VZV pathogenesis in vivo.Varicella-zoster virus (VZV) is a human ␣-herpesvirus that causes chickenpox and herpes zoster (shingles). The ␣-herpesviruses contain two highly conserved genes that are predicted to encode protein kinases by sequence homology to eukaryotic serine͞threonine kinases (1, 2). In VZV, these genes are encoded by ORF47, in the unique, long region of the genome, and by ORF66, in the unique, short region. ORF47 encodes a 54-kDa phosphoprotein found in the cytoplasm and nucleus of infected cells and in the virion capsid͞tegument fraction. ORF66 kinase is a 48-kDa phosphoprotein located only in the cytoplasm (3). Homologs of ORF47 are found in ␣-, -and ␥-herpesviruses, but ORF66 homologs are specific to the ␣-herpesviruses (1, 2, 4-9).The ORF47 putative kinase phosphorylates itself and ORF62, the major immediate early (IE) transactivator, uses both ATP and GTP as phosphate donors (10, 11). The other VZV IE proteins encoded by ORF4, ORF61, and ORF63, and the major glycoprotein E (gE), are not phosphorylated by ORF47 kinase in vitro (3,(10)(11)(12). VZV ORF47, like related kinases, is dispensable for replication in vitro (6,(12)(13)(14). The HSV-1 UL13 kinase is autophosphorylated and necessary for the posttranslational modification of HSV-1 regulatory proteins infected cell protein (ICP)22, homolog of VZV ORF63, and ICP0, homolog of VZV ORF61 but not ICP4, the ORF62 homolog (13,(15)(16)(17). The HSV-1 UL13 gene also is required for the virion host shutoff effect (14). More recently, UL13 kinase has been shown to phosphorylate the HSV-1 glycoprotein E͞I Fc receptor complex and the cellular protein elongation factor 1␦ (18,19).Like the ORF47 protein, the ORF66 putative kinase is not required for phosphorylation of the IE genes and is not essential in tissue culture. Although neither muta...
In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer. Varicella-zoster virus (VZV), the causative agent of chicken pox (varicella) and shingles (herpes zoster), is a highly cellassociated herpesvirus both in vitro and in vivo (7, 23). Similar to the situation with a closely related alphaherpesvirus, Marek's disease virus (MDV), VZV spreads directly from cell to cell, presumably utilizing the machinery involved in adherens junction modeling and architecture. Infectious virus is released into the environment only after lytic VZV replication in the skin and respiratory mucous membranes of infected individuals (2, 43). Efficient and timely coordinated interaction between the tegument, a proteinaceous structure surrounding the icosahedral nucleocapsid, and envelope membrane (glyco) proteins plays a crucial role in the secondary envelopment and spread of herpesviruses in general and VZV in particular (35). It has been shown for related viruses, such as herpes simplex virus, that the inner layer of tegument is tightly associated with the nucleocapsid, whereas the outer layer of tegument provides the link to envelope proteins (34, 36). The current model of herpesviral tegumentation predicts that the inner layer of tegument is added to the nucleocapsid in the nucleus and deenveloped particles in the cytoplasm, while proteins representing the outer layer are thought to accumulate at cytoplasmic membranes where they make contact with their respective partners, either other tegument proteins or membrane proteins or both (34-36). Through these intricately regulated interactions, final secondary envelopment of particles is facilitated and, ultimately, infectious virus is produced and released.Determination of the role of individual tegument or membrane (glyco)proteins in the replication cycle of various herpesviru...
To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) Human cytomegalovirus (HCMV), a member of the herpesvirus family, is an important pathogen implicated in a variety of diseases in newborn and immunocompromised individuals (for reviews, see references 2 and 59). Like other members of the herpesvirus family, the genome of HCMV is temporally expressed during the viral life cycle (16, 56,81,88,89 (16,17,29,33,34, 56,64,79,80,82,88,89,91). At least three IE RNAs have been found to be transcribed from these regions (Fig. 1B) (62,66,77,80).In previous studies, we and others have used transientexpression assays to demonstrate that HCMV early promoters as well as heterologous viral and cellular promoters can be activated by the region of the genome specifying the IE1 and IE2 gene products (4,5,9,12,15,18,20,23,26,29,39,53,60,63,67,75,85,87). Recent studies suggest that the IE2 86-kDa protein plays a major role in activating HCMV early promoters as well as in repressing its own promoter (the major IE promoter), while the IE1 72-kDa protein acts to enhance the activity of the major IE promoter and may augment the stimulatory effect of the IE2 86-kDa protein (10-12, 26, 28, 50, 53, 61-63, 69, 78).Any consideration of the mechanism by which the HCMV IE1 and IE2 proteins affect transcription must take into account the fact that multiple viral and cellular promoters 1238 on April 27, 2019 by guest
The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.
The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-␥. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells. Varicella-zoster virus (VZV)is an alphaherpesvirus that causes chicken pox, or varicella, establishes lifelong latency in the sensory ganglia, and later reactivates to cause shingles, or herpes zoster. The pathogenesis of primary VZV infection is characterized by inoculation of respiratory mucosa, followed by a cell-associated viremia and a subsequent vesicular rash that develops 10 to 21 days after exposure (3). T cells appear to be a major target cell for VZV viremia. The virus infects primary human T cells in vitro and exhibits tropism for T cells in thymus/liver xenografts in the severe combined immunodeficiency (SCID)-hu mouse model in vivo (28,35,49). VZV alters cellular gene expression in T cells, as shown by downregulation of major histocompatibility (MHC) class I protein expression and microarray analysis of gene transcription (1, 18). Importantly, T cells have the capacity to transport infectious VZV through the circulation, resulting in the formation of typical cutaneous lesions in SCID-hu skin xenografts (29). The goal of these experiments was to further investigate the T-cell tropism of VZV by examining virion formation, effects on apoptotic and interferon (IFN) pathways in human T cells, and the contributions of the ORF66 protein to these processes, based on previous evidence that preventing ORF66 protein expression decreased VZV virulence in T-cell xenografts in vivo (37).The putative early gene ORF66 encodes a 47-kDa protein that localizes to both nuclei and cytoplasm of infected cells in vitro and is present in the VZV virion (22,50). Sequence analysis has revealed that ORF66 is highly homologous to serine/threonine protein kinases in other...
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