Differential Role for Host Translation Factors in Host and Viral Protein Synthesis during Human Cytomegalovirus Infection

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Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. To facilitate viral spread, we have previously shown HCMV to extend the short 48-h life span of monocytes. Mechanistically, HCMV upregulated two specific cellular antiapoptotic proteins, myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of fully active caspase 3 and the subsequent initiation of apoptosis. We now show that HCMV more robustly upregulated Mcl-1 than normal myeloid growth factors and that Mcl-1 was the only myeloid survival factor to rapidly induce HSP27 prior to the 48-h cell fate checkpoint. We determined that HCMV glycoproteins gB and gH signal through the cellular epidermal growth factor receptor (EGFR) and ␣v␤3 integrin, respectively, during viral entry in order to drive the increase of Mcl-1 and HSP27 in an Akt-dependent manner. Although Akt is known to regulate protein stability and transcription, we found that gB-and gH-initiated signaling preferentially and cooperatively stimulated the synthesis of Mcl-1 and HSP27 through mTOR-mediated translation. Overall, these data suggest that the unique signaling network generated during the viral entry process stimulates the upregulation of select antiapoptotic proteins allowing for the differentiation of short-lived monocytes into long-lived macrophages, a key step in the viral dissemination strategy. H uman cytomegalovirus (HCMV), a betaherpesvirus, is highly prevalent throughout the United States, with 50 to 80% of the population being seropositive (1). HCMV infection is generally asymptomatic in immunocompetent individuals although infection has been linked to chronic inflammatory diseases, such as atherosclerosis and acute infection mononucleosis (2, 3). In contrast, HCMV infection causes significant morbidity and mortality in immunocompromised individuals, including AIDS patients, neonates, and transplant recipients (4-6). In these patients, HCMV diseases are diverse with respect to the organ site, including eyes (retinitis), brain (encephalopathy), central nervous system (polyradiculopathy), lungs (pneumonitis), and gastrointestinal tract (gastroenteritis), which can lead to systemic organ failure (7,8). The systemic pathogenesis established by HCMV is dependent on the spread of the virus from the initial point of infection to peripheral organs. IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic within the human population. Although primary infection is generally asymptomatic in immunocompetent individualsMonocytes are believed to be the primary cell type responsible for the systemic dissemination of HCMV during both symptomatic and asymptomatic infections (9-11). However, monocytes have a limited life span of 48 h following release from the bone marrow, after which these cells either undergo apoptosis or differentiate into long-lived macrophages in the presence of a differentiation stimulus (1...

Section: Methodsmentioning

confidence: 99%

Human Cytomegalovirus Stimulates the Synthesis of Select Akt-Dependent Antiapoptotic Proteins during Viral Entry To Promote Survival of Infected Monocytes

Peppenelli

Arend

Cojohari

et al. 2016

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“…Unless otherwise noted, all infections were performed at a multiplicity of infection of 1 in 300 l of medium. The amount of cell-free virus in the supernatant was quantified by the TCID 50 method on MRC-5 fibroblasts as previously described (50).…”

Section: Methodsmentioning

confidence: 99%

Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication

Ziehr

Vincent

Moorman

2016

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Human cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2␣ were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2␣ phosphorylation. Depletion of PKR prevented eIF2␣ phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication. IMPORTANCETo successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease. Human cytomegalovirus (HCMV), like all viruses, requires host ribosomes and translation factors for the synthesis of viral proteins. Consequently, upon sensing infection, host antiviral defenses inactivate critical translation factors, leading to reduced viral replication. To circumvent these defenses, HCMV manipulates antiviral signaling pathways to allow for efficient viral protein synthesis. Thus, the interface of HCMV with the host translation machinery lies at the front line of the battle between host and virus for control of the infected cell.Perhaps the best-studied antiviral defense targeting viral mRNA translation is the RNA-dependent protein kinase R (PKR). PKR binds to double-stranded RNAs (dsRNAs) produced during viral infections, resulting in PKR dimerization and activating autophosphorylation (1-4). Activated PKR in turn inhibits mRNA translation by phosphorylating its substrate the eukaryotic initiation factor 2 alpha (eIF2␣) (5-8). eIF2␣ plays a critical ...

“…Total RNA was extracted using TRIzol according to the manufacturer's directions as described previously (47,48). Briefly, cell pellets were resuspended in TRIzol and extracted with chloroform, and RNA was precipitated with isopropanol.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…5= rapid amplification of cDNA ends (5= RACE) was performed using the RLM-RACE (Ambion) kit according to the manufacturer's protocol as previously described (47). Briefly, total or polysome-associated RNA was treated with calf intestinal phosphatase (CIP) to dephosphorylate uncapped or degraded RNAs.…”

Section: Cells and Virusesmentioning

confidence: 99%

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Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection

Arend

Ziehr

Vincent

et al. 2016

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Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5= untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection. IMPORTANCEThe HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells. The human cytomegalovirus (HCMV) IE1 and IE2 proteins are critical regulators of the viral replicative cycle. Both proteins are immediately expressed upon infection and together stimulate the expression of host and viral genes necessary for virus replication (1). IE2 acts as a general transcription factor that broadly transactivates host genes and viral early and late genes to facilitate virus replication (2-12). IE1 promotes transcription from the HCMV genome by inhibiting histone deactylases (HDACs) (13-15), which otherwise limit virus transcription by forming inhibitory chromatin structures on the viral genome. Reexpression of IE1 and IE2 is also thought to be critical for the reactivation of quiescent HCMV genomes from latent infection. Thus, understanding the regulatory mechanisms controlling IE1 and IE2 expression is im...