Cytomegalovirus Destruction of Focal Adhesions Revealed in a High-Throughput Western Blot Analysis of Cellular Protein Expression

Abstract

Human cytomegalovirus (HCMV) is a clinically important herpesvirus associated with severe disease following congenital infection and in immunocompromised individuals. As a herpesvirus, primary infection is accompanied by lifelong persistence during which the infection must be controlled by continuous host immune surveillance. HCMV has the largest genome of any characterized human virus (ϳ236 kb) and is predicted to encode on the order of 165 potential open reading frames (15). Systematic deletion of individual open reading frames from the Towne strain revealed that only 45 are essential for virus replication in vitro; thus, accessory genes account for most of the HCMV coding capacity (16). While the functions of most of these genes have not been determined, a significant number have been implicated in the targeting of both the innate and adaptive host immune responses (40). HCMV has also recently been shown to express microRNAs during productive infection that also have the potential to modulate host cell gene expression (17,21,43). A high-throughput proteomic approach was used in this study with a view to providing further insight into how the virus may modulate its host cell.HCMV replicates slowly in permissive human fibroblasts, with gene expression conventionally being divided into three phases: immediate-early (IE), early, and late (reviewed in reference 40). Early-phase transcription precedes viral DNA replication (initiates at 16 to 24 h postinfection [hpi]), with significant virus production detected from 72 hpi and peaking at approximately 144 hpi. Productive HCMV infection is associated with an overall stimulation of both cellular transcription and translation (50). Virion binding (13, 59), release of virion tegument proteins following virion fusion (6, 36), and de novo expression of powerful transcriptional regulators (most notably, IE2 and IE1) during infection all modulate cellular gene expression. Following infection, while cellular proteins associated with DNA metabolism are induced, host cell cyclins are dysregulated and licensing of host cell DNA replication is inhibited, resulting in a "pseudo-G 1 " environment compatible with efficient virus DNA replication (3,5,25). Microarray experiments have proved highly informative in revealing the dynamic regulation of steady-state levels of cellular transcripts, both positively and negatively, during the course of infection (7,26,48,65,66). HCMV is known not only to regulate transcriptional initiation but also to control RNA processing (1,9,14,20,35,63), translation, and posttranslational modification and protein trafficking (31,34,44,54,61). The result of many of these processes is liable to be an alteration of host cell protein levels.

Human cytomegalovirus pUL37x1-induced calcium flux activates PKCα, inducing altered cell shape and accumulation of cytoplasmic vesicles

“…3B). Consistent with an earlier report (18), WT virus also substantially reduced cell surface staining with vinculin (Fig. 3A, Upper), a protein found at the intersection of actin bundles and membrane attachment sites (19).…”

Upregulation of Nrf2 expression by human cytomegalovirus infection protects host cells from oxidative stress

“…HCMV has been shown to increase hsp90 expression, and our data indicate that HCMV-mediated Nrf2 activation was abolished following treatment with CK2 inhibitors (Stanton et al, 2007). Altogether, these data would suggest that HCMV activates Nrf2 by manipulating the hsp90 and Keap1 interaction.…”

“…HCMV infection prevented the nuclear translocation of Nrf2 and failed to activate HO-1 expression. However, other investigations have shown that HO-1 expression is increased upon HCMV infection at the RNA or protein level when primary fibroblasts are used (Browne et al, 2001;Stanton et al, 2007). Therefore, the effect of HCMV infection on the Nrf2 pathway needs to be further clarified.…”

Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

“…HCMV strain Merlin (p5) (27) was utilized to generate BACs as described below. Infections were performed as described previously (78), and viruses were titrated in triplicate by plaque assay for 14 days on HFFFs using a 1% Avicel overlay (79). Cultures that exhibited small plaques at 14 days PT were incubated for a further 14 days and recounted.…”