Human Cytomegalovirus Tegument Protein pp71 Directs Long-Term Gene Expression from Quiescent Herpes Simplex Virus Genomes

Abstract

The human cytomegalovirus tegument protein pp71 is important for transactivation of immediate-early (IE) gene expression and for the efficient initiation of virus replication. We have analyzed the properties of pp71 by assaying its effects on gene expression from the genome of in1312, a herpes simplex virus type 1 (HSV-1) mutant devoid of functional VP16, ICP0, and ICP4. Upon infection of human fibroblasts, in1312-derived viruses are repressed and retained in a quiescent state, but the presence of pp71 prevented the quiescent state from being attained. Reporter gene cassettes cloned into the in1312 genome, in addition to the endogenous IE promoters, remained active for at least 12 days postinfection, and infected cells were viable and morphologically normal. Cells expressing pp71 remained responsive to the HSV-1 transactivating factors VP16 and ICP4 and to trichostatin A. The C-terminal 61 amino acids, but not the LACSD motif, were required for pp71 activity. In addition to preventing attainment of quiescence, pp71 was able to disrupt the quiescent state of in1312 derivatives and promote the resumption of viral gene expression after a lag of approximately 3 days. The results extend the functional analysis of pp71 and suggest a degree of similarity with the HSV-1 IE protein ICP0. The ability to provoke slow reactivation of quiescent genomes, in conjunction with cell survival, represents a novel property for a viral structural protein.Many herpesviruses utilize virion proteins as transactivators of immediate-early (IE) gene expression to ensure the rapid initiation of productive replication. In the case of herpes simplex virus type 1 (HSV-1), the well-studied tegument protein VP16 activates IE transcription through specific TAATGA RAT elements that are present in all IE promoters (6, 43). For human cytomegalovirus (HCMV), the 559-amino-acid tegument phosphoprotein pp71, encoded by the gene UL82 (9, 42, 51), fulfills an equivalent role, although the mode of action of this protein is not understood in detail.The transactivating properties of pp71 were first recognized in cotransfection assays. Expression from cytomegalovirus IE promoters on plasmid templates was stimulated by the presence of a plasmid that expressed pp71, and the effect was mediated by sequence elements that bind the cellular transcription factors ATF or AP-1 (33). Dependence on ATF recognition sites was also observed in cotransfection studies that investigated the response of the HCMV US11 promoter in the presence of pp71, although in this case the IE protein IE86 was also present (7). The promoter for intercellular adhesion molecule 1 was activated by pp71, in conjunction with IE86, via Sp1 recognition sites (32). Limited stimulation of endogenous intercellular adhesion molecule 1 expression by pp71 alone was also reported in these studies. When HCMV virion DNA was delivered to cells by transfection, the addition of plasmids that expressed pp71 increased the numbers of progeny plaques and the rate of progress of infection (3). This effect could no...

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity

“…In one approach to achieve this goal, ICP0 has been substituted with similar IE genes from other herpesviruses, including the pp71 gene of human cytomegalovirus (HCMV) (33,34), a betaherpesvirus, with the notion that the products of these genes may provide the antisilencing functions of ICP0 without toxicity (35). However, it is well-known that pp71 degrades a chromatin-remodeling factor, hDaxx, followed by disruption of the hDaxx-ATRX complex (36,37) and members of the retinoblastoma tumor-suppressor family of proteins (38).…”

Reactivation of Expression from Quiescent Herpes Simplex Virus Type 1 Genomes in the Absence of Immediate-Early Protein ICP0

“…This procedure results in a plasmid with YFP controlled by the HCMV major IE promoter (MIEP) and the simian virus 40 polyadenylation sequences, all embedded in the HSV-1 thymidine kinase (TK) coding region. Plasmid pCP376 has the Escherichia coli lacZ region, controlled by the HCMV MIEP, inserted into the coding sequences of the nonessential UL43 open reading frame, as described previously (37). Plasmid pMC17 consists of the HSV-1 VP16 coding region cloned into pUC9 (1).…”

“…Mutants derived from in1814 and additionally impaired for the expression of ICP0 and ICP4 were used to obtain cultures in which most cells contained at least one quiescent genome (14,37,38). Even greater reduction in cytotoxicity was achieved by inactivating all IE genes, resulting in mutants that established quiescent infections in human fibroblasts and Vero cells (21,23,43).…”

“…Mutant KM110 lacks the ICP0 coding region and the C-terminal activating domain of VP16, and although potentially capable of replication, this mutant is sufficiently impaired to establish quiescent infection at high efficiency in human fibroblasts without cytotoxicity (34). In each of these systems, provision of ICP0 reactivated the quiescent genome (21,31,37,38,43).…”

Promyelocytic Leukemia-Nuclear Body Proteins: Herpesvirus Enemies, Accomplices, or Both?

“…The exogenous expression of ICP0 or pp71 can activate gene expression from quiescent HSV-1 genomes [98][99][100][101]. Furthermore, latently-infected cells express antisense transcripts that may affect the expression of ICP0 [102][103][104][105] or pp71 [106], implying that repressing the expression of these proteins may be important for the maintenance of the latent state.…”