Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, Toledo BAC virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. Toledo BAC , like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (Toledo BAC ) retained its virulence. The 15-kb segment was then deleted from Toledo BAC , and the resulting virus, Toledo ⌬15kb , was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. Toledo ⌬15kb had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the Toledo ⌬15kb genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.

In its course of human infection, varicella-zoster virus (VZV) infects rarely dividing cells such as dermal fibroblasts, differentiated keratinocytes, mature T cells, and neurons, none of which are actively synthesizing DNA; however, VZV is able to productively infect them and use their machinery to replicate the viral genome. We hypothesized that VZV alters the intracellular environment to favor viral replication by dysregulating cell cycle proteins and kinases. Cyclin-dependent kinases (CDKs) and cyclins displayed a highly unusual profile in VZV-infected confluent fibroblasts: total amounts of CDK1, CDK2, cyclin B1, cyclin D3, and cyclin A protein increased, and kinase activities of CDK2, CDK4, and cyclin B1 were strongly and simultaneously induced. Cyclins B1 and D3 increased as early as 24 h after infection, concurrent with VZV protein synthesis. Confocal microscopy indicated that cyclin D3 overexpression was limited to areas of IE62 production, whereas cyclin B1 expression was irregular across the VZV plaque. Downstream substrates of CDKs, including pRb, p107, and GM130, did not show phosphorylation by immunoblotting, and p21 and p27 protein levels were increased following infection. Finally, although the complement of cyclin expression and high CDK activity indicated a progression through the S and G 2 phases of the cell cycle, DNA staining and flow cytometry indicated a possible G 1 /S blockade in infected cells. These data support earlier studies showing that pharmacological CDK inhibitors can inhibit VZV replication in cultured cells.Varicella-zoster virus (VZV) is a member of the alphaherpesvirus family, causing chicken pox (varicella) upon primary infection and shingles (zoster) after reactivation from latency in ganglia. VZV shows a tropism for rarely dividing cell types, including differentiated keratinocytes, dermal fibroblasts, epithelium, neurons, and memory T cells (5). These cell types are typically quiescent in their in vivo states, yet VZV productively infects them all. How this large DNA virus is able to replicate its genome in an environment inhospitable to duplication is poorly understood. Less is known about the interaction of VZV with host cells compared to other alphaherpesviruses, because it is tightly cell-associated in culture and synchronous, high-multiplicity infections are not feasible.The involvement of cellular kinases in VZV replication has recently become a topic of interest. Casein kinases I and II (CK-I and CK-II) have been found to phosphorylate the VZV glycoprotein gE, which is hypothesized to aid in cell-to-cell spread of progeny (23, 30). Glycoprotein gI was also shown to be phosphorylated by cyclin-dependent kinase 1 (CDK1) in vitro, and this phosphorylation was blocked by roscovitine, a specific CDK inhibitor (57). Our group has previously shown that roscovitine prevented VZV replication in cultured cells; however, the inhibition of glycoprotein phosphorylation was not the only mechanism of action, since early and late gene expression were also blocked (51).CDKs are al...

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Varicella-zoster virus (VZV), an alphaherpesvirus restricted to humans, infects differentiated cells in vivo,including T lymphocytes, keratinocytes, and neurons, and spreads rapidly in confluent cultured dermal fibroblasts (HFFs). In VZV-infected HFFs, atypical expression of cyclins D3 and B1 occurs along with the induction of cyclin-dependent kinase (CDK) activity. A specific CDK1 inhibitor blocked VZV spread, indicating an important function for this cellular kinase in VZV replication. CDK activity assays of infected cells revealed a large viral phosphoprotein that was identified as being the major immediate-early transactivator, IE62. Since IE62 colocalized with CDK1/cyclin B1 by confocal microscopy, we investigated whether this cellular kinase complex interacts with IE62. Using recombinant fragments of IE62 spanning the entire amino acid sequence, we found that purified CDK1/cyclin B1 phosphorylated IE62 at residues T10, S245, and T680 in vitro. Immunoprecipitation of cyclin B1 from VZV-infected HFFs indicated that IE62 was included in the complex within infected cells. The full-length IE62 protein, obtained by immunoprecipitation from infected cells, was also phosphorylated by purified CDK1/cyclin B1. Based on IE62/CDK1/cyclin B1 colocalization near viral assembly regions, we hypothesized that these cellular proteins could be incorporated into VZV virions with IE62. Purified virions were analyzed by immunoblotting for the presence of CDK1 and cyclin B1, and active CDK1 and cyclin B1 were present in the VZV tegument with IE62 and were sensitive to detergent treatment. Thus, IE62 is a substrate for CDK1/cyclin B1, and virions could deliver the active cellular kinase to nondividing cells that normally do not express it.Varicella-zoster virus (VZV) (human herpesvirus type 3) infects epithelia, skin, T cells, and neurons and causes the human diseases chickenpox (varicella) and shingles (herpes zoster). The 125-kbp double-stranded viral DNA genome encodes 69 open reading frames (ORFs); this large proteome mediates essential viral functions and manipulates the cellular environment. The major immediate-early transactivator protein, IE62, is encoded by duplicated ORFs 62 and 71 and, as a tegument protein, is delivered to newly infected cell nuclei, where it initiates VZV replication by transactivating viral immediate-early and early genes (23,25). While IE62 binds several viral proteins such as ORF4 (49), ORF9 (7), ORF47 (37), and ORF63 (31) and cellular transcription factors including upstream stimulatory factor, TATA-binding protein, and Sp1 (43-45), there is no information regarding interactions with cyclin-dependent kinases (CDKs). Roscovitine, a CDK inhibitor, blocks VZV replication in cell culture and in an ex vivo skin model, but it is not clear how CDK activity benefits the virus (51, 52).VZV encodes two viral kinases, ORF47 and ORF66, both serine/threonine kinases that phosphorylate IE62 (10, 37). ORF66 phosphorylation of IE62 is responsible for the nuclearcytoplasmic shuttling of this protein later in infe...