Human Cytomegalovirus Immediate-early Transcription Research Articles

Human cytomegalovirus reprograms hematopoietic progenitor cells into immunosuppressive monocyte to achieve latency

Abstract The precise cell type hosting latent human cytomegalovirus (HCMV) remains elusive. Here we report that HCMV reprograms human hematopoietic progenitor cells (HPCs) into a unique monocyte subset to achieve latency. Unlike conventional monocytes, this monocyte subset possesses higher levels of B7-H4, IL-10 and iNOS, longer lifespan and strong immunosuppressive capacity. Cell sorting of peripheral blood from latently infected human donors confirms that only this monocyte subset, representing less than 0.1% of peripheral mononuclear cells, is HCMV genome-positive but immediate-early (IE)-negative. Mechanistic studies demonstrate that HCMV promotes the differentiation of HPCs into this monocyte subset by activating cellular signal transducer and activator of transcription 3 (STAT3). In turn, this monocyte subset generates high level of nitric oxide (NO) to silence HCMV IE transcription and promote viral latency. By contrast, US28-knockout HCMV mutant incapable of activating STAT3, fails to reprogram the HPCs and achieve latency. Our findings reveal that HCMV differentiates human HPCs into long-life, immunosuppressive monocyte subset for viral latency via activating STAT3/iNOS/NO axis.

Human Cytomegalovirus Protein pp71 Displaces the Chromatin-Associated Factor ATRX from Nuclear Domain 10 at Early Stages of Infection

The human cytomegalovirus (HCMV) tegument protein pp71, encoded by gene UL82, stimulates viral immediate-early (IE) transcription. pp71 interacts with the cellular protein hDaxx at nuclear domain 10 (ND10) sites, resulting in the reversal of hDaxx-mediated repression of viral transcription. We demonstrate that pp71 displaces an hDaxx-binding protein, ATRX, from ND10 prior to any detectable effects on hDaxx itself and that this event contributes to the role of pp71 in alleviating repression. Introduction of pp71 into cells by transfection, infection with a pp71-expressing herpes simplex virus type 1 vector, or by generation of transformed cell lines promoted the rapid relocation of ATRX from ND10 to the nucleoplasm without alteration of hDaxx levels or localization. A pp71 mutant protein unable to interact with hDaxx did not affect the intranuclear distribution of ATRX. Infection with HCMV at a high multiplicity of infection resulted in rapid displacement of ATRX from ND10, the effect being observed maximally by 2 h after adsorption, whereas infection with the UL82-null HCMV mutant ADsubUL82 did not affect ATRX localization even at 7 h postinfection. Cell lines depleted of ATRX by transduction with shRNA-expressing lentiviruses supported increased IE gene expression and virus replication after infection with ADsubUL82, demonstrating that ATRX has a role in repressing IE transcription. The results show that ATRX, in addition to hDaxx, is a component of cellular intrinsic defenses that limit HCMV IE transcription and that displacement of ATRX from ND10 by pp71 is important for the efficient initiation of viral gene expression.

Role of the cellular protein hDaxx in human cytomegalovirus immediate-early gene expression

Human cytomegalovirus (HCMV) immediate-early (IE) transcription is stimulated by virion phosphoprotein pp71, the product of gene UL82. It has previously been shown that pp71 interacts with the cellular protein hDaxx and, in the studies presented here, the significance of this interaction was investigated for HCMV IE gene expression. In co-transfection experiments, the presence of hDaxx increased the transcriptional response of the HCMV major IE promoter (MIEP) to pp71, but it was not possible to determine whether the effect was due to an interaction between the two proteins or to stimulation of hDaxx synthesis by pp71. The use of small interfering RNA (siRNA) in long- and short-term transfection approaches reduced intracellular hDaxx levels to no more than 3 % of normal. Infection of hDaxx-depleted cells with herpes simplex virus recombinants containing the HCMV MIEP revealed significantly greater promoter activity when hDaxx levels were minimal. Similarly, reducing intracellular hDaxx amounts resulted in greater IE gene expression during infection with an HCMV mutant lacking pp71, but had no effect on IE transcription during infection with wild-type HCMV. The results suggest that hDaxx is not important as a positive-acting factor for the stimulation of HCMV IE transcription by pp71. Instead, it appears that hDaxx acts as a repressor of IE gene expression, and it is proposed here that the interaction of pp71 with hDaxx is important to relieve repression and permit efficient initiation of productive replication.

Infection with human cytomegalovirus enhances bacterial adhesiveness and invasiveness in permissive and semipermissive cells.

The effect of human cytomegalovirus (HCMV) infection on adhesiveness and invasiveness of Salmonella typhimurium was examined in cells permissive (human embryo fibroblasts (HE)), semipermissive (A549) and nonpermissive (HEp-2) for the virus. Preinfection of the cells with HCMV induced enhanced adhesiveness and invasiveness of bacteria in the permissive HE cells. In the semipermissive A549 cells, where HCMV immediate-early (IE) mRNA transcripts and IE proteins were detected, a significant effect on the initial phase of invasiveness, the adherence phase, was demonstrated. HCMV had no effect on invasiveness of S. typhimurium in nonpermissive HEp-2 cells. Neither HCMV IE transcripts nor IE proteins could be detected in these cells.

Cellular oncogene activation by human cytomegalovirus Lack of correlation with virus infectivity and immediate early gene expression

The contribution of expression of human cytomegalovirus (HCMV) immediate early (IE) genes to the rapid and transient increase in cellular (c)-oncogene (fos, jun, myc) transcription following HCMV infection was investigated. A partial temporal overlap was observed between the increases in c-oncogene RNA levels and the increase in either transcripts from HCMV IE genes or the number of cells in which HCMV IE proteins were detected. The increases in c-oncogene RNA levels, however, slightly preceded the increase in the detection of HCMV IE transcripts or proteins. To distinguish between the temporal coincidence and a direct relationship between expression of HCMV IE genes and the increased transcription of c-oncogenes, the number of cells synthesizing HCMV IE proteins was reduced by infecting with virus stock enriched in defective particles. Alternatively, the synthesis of HCMV IE proteins was essentially eliminated by ultra-violet (UV) irradiation of virus stock or by inhibitors of protein synthesis. Virus stocks enriched in defective particles demonstrated a substantially reduced capacity to direct the synthesis of HCMV IE proteins, but were more efficient in activating c-oncogene expression than infectious virus stocks. Elimination of expression of HCMV IE genes by UV-irradiation of virus stock or by inhibiting de novo viral and/or cellular protein synthesis with cycloheximide (100 micrograms/ml) or anisomycin (100 micrograms/ml) did not eliminate the HCMV-induced increase in RNA levels of c-oncogenes. These data indicate that activation of these early response cellular genes is independent from de novo expression of HCMV IE proteins, and possibly involves biologically active virion proteins that are related to the induction of a cascade of cellular events associated with the binding of HCMV to its cellular receptor.

Transcriptional activation of the major immediate early transcription unit of human cytomegalovirus by heat-shock, arsenite and protein synthesis inhibitors.

In Rat-9G cells several copies of the major immediate early (IE) transcription unit (regions 1 and 2) of the human cytomegalovirus (HCMV) are stably integrated. The cells show a repressed phenotype for IE expression but can be induced by inhibition of protein synthesis. In this report we present evidence that the repressed phenotype is due to the absence of IE transcription and that heat-shock and sodium arsenite treatments each result in the transcriptional activation of the repressed IE transcription unit. Either treatment resulted in the induction of HCMV IE transcripts and IE nuclear antigen expression. An octameric DNA sequence present in three of the 18 bp IE enhancer elements (GGACTTTC) resembles the cellular heat-shock element core consensus sequence and may therefore be involved in the heat-shock response.