The Human Cytomegalovirus UL36 Gene Controls Caspase-Dependent and -Independent Cell Death Programs Activated by Infection of Monocytes Differentiating to Macrophages

115

159

Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractablein vitromodel system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractablein vitromodel system with which to study HCMV latency and reactivation.

mentioning

confidence: 99%

A Myeloid Progenitor Cell Line Capable of Supporting Human Cytomegalovirus Latency and Reactivation, Resulting in Infectious Progeny

O’Connor

Murphy

2012

115

159

“…Only a few loci (UL36 to -38, UL115 to -119, UL122 to -123, US3, and IRS1/TRS1) within the ϳ240-kbp genome of HCMV are transcribed at IE times of infection, but alternative RNA splicing and translation initiation increase the diversity of the resulting gene products. To facilitate later phases of infection, IE proteins impair many cellular functions, including apoptosis (18,43,44,62), cellular DNA synthesis (48, 72), STAT signaling (51), protein kinase R activity (10, 42), and major histocompatibility complex (MHC) class I-mediated antigen presentation (2, 31). Furthermore, IE proteins are responsible for the activation of viral early genes that encode proteins required for viral DNA replication (70).…”

mentioning

confidence: 99%

Inhibition of Human Cytomegalovirus Immediate-Early Gene Expression by Cyclin A2-Dependent Kinase Activity

Oduro

Uecker

Hagemeier

et al. 2012

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G 0 /G 1 phase of the cell division cycle. S/G 2 cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G 2 -specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G 1 was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.

“…To extend cell survival during its protracted lytic replication, HCMV encodes several antiapoptotic products, including the UL37, UL36, UL38, and major immediate-early 1/2 (IE1/2) gene products (24,25,49,77,85,93) as well as the ␤2.7 RNA, which inhibits proapoptotic signals from the mitochondrial GRIM complex (66).…”

mentioning

confidence: 99%

The Human Cytomegalovirus Protein UL37 Exon 1 Associates with Internal Lipid Rafts

Williamson

Zhang

Colberg-Poley

2011

The human cytomegalovirus (HCMV) protein UL37 exon 1 (pUL37x1), also known as viral mitochondrionlocalized inhibitor of apoptosis (vMIA), sequentially traffics from the endoplasmic reticulum (ER) through mitochondrion-associated membranes (MAMs) to the outer mitochondrial membrane (OMM), where it robustly inhibits apoptosis. Here, we report the association of pUL37x1/vMIA with internal lipid rafts (LRs) in the ER/MAM. The MAM, which serves as a site for lipid transfer and calcium signaling to mitochondria, is enriched in detergent-resistant membrane (DRM)-forming lipids, including cholesterol and ceramide, which are found in lower concentrations in the bulk ER. Sigma 1 receptor (Sig-1R), a MAM chaperone affecting calcium signaling to mitochondria, is anchored in the MAM by its LR association. Because of its trafficking through the MAM and partial colocalization with Sig-1R, we tested whether pUL37x1/vMIA associates with MAM LRs. Extraction with methyl-␤-cyclodextrin (M␤CD) removed pUL37x1/vMIA from lysed but not intact cells, indicating its association with internal LRs. Furthermore, the isolation of DRMs from purified intracellular organelles independently verified the localization of pUL37x1/vMIA within ER/MAM LRs. However, pUL37x1/vMIA was not detected in DRMs from mitochondria. pUL37x1/vMIA associated with LRs during all temporal phases of HCMV infection, indicating the likely importance of this location for HCMV growth. Although detected during its sequential trafficking to the OMM, the pUL37x1/vMIA LR association was independent of its mitochondrial targeting signals. Rather, it was dependent upon cholesterol binding. These studies suggest a conserved ability of UL37 proteins to interact with cholesterol and LRs, which is functionally distinguishable from their sequential trafficking to mitochondria.Human cytomegalovirus (CMV) (HCMV) is a medically important herpesvirus which causes serious diseases in congenitally infected infants and in immunocompromised individuals (54). To extend cell survival during its protracted lytic replication, HCMV encodes several antiapoptotic products, including the UL37, UL36, UL38, and major immediate-early 1/2 (IE1/2) gene products (24,25,49,77,85,93) as well as the ␤2.7 RNA, which inhibits proapoptotic signals from the mitochondrial GRIM complex (66).The HCMV UL37 IE product, the UL37 exon 1 (UL37x1) protein (pUL37x1), also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), sequesters proapoptotic Bax at the outer mitochondrial membrane (OMM) and prevents cytochrome c release and the subsequent initiation of the proapoptotic cascade (3, 58, 60, 62). pUL37x1/vMIA also causes calcium (Ca 2ϩ ) efflux from endoplasmic reticulum (ER) stores and F-actin cytoskeleton disruption, which results in early and late cytopathologies (63,72). pUL37x1/vMIA also transactivates the expression of HCMV early genes whose products are required for its genome replication (5,14,67,90). At late times of infection, pUL37x1/vMIA further extends cell viability by inhibiting a mitoc...