Cytomegalovirus miRNAs Target Secretory Pathway Genes to Facilitate Formation of the Virion Assembly Compartment and Reduce Cytokine Secretion

Hook, Lauren M.; Grey, Finn; Grabski, Robert; Tirabassi, Rebecca S.; Doyle, Tracy J.; Hancock, Meaghan H.; Landais, Igor; Jeng, Sophia; McWeeney, Shannon K.; Britt, William J.; Nelson, Jay A.

doi:10.1016/j.chom.2014.02.004

Cited by 123 publications

(171 citation statements)

References 45 publications

(67 reference statements)

Supporting

Mentioning

161

Contrasting

Unclassified

Order By: Relevance

“…cVAC biogenesis may thus be a product of the virus activating, and possibly modifying, a previously programmed biological process. Like other biological pathways regulated by HCMV (e.g., immune responses, apoptosis, and the AKT/PI3K/mTOR pathway) (35,36), we anticipate multiple points of control of the process by the virus, including the recently described role of HCMV microRNAs in cVAC biogenesis (25). In addition to the virologic lessons, elucidation of this pathway and its points of control will be informative with respect to important areas of cell biology.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, an HCMV locus spanning UL133 to UL138 is required for cVAC development in human microvascular endothelial cells, but not in embryonic lung fibroblasts (24), indicating that cell-specific viral factors can be involved. (vii) In addition to protein regulators of cVAC biogenesis, the HCMV microRNAs (miRNAs) miR UL112-1, US5-1, and US5-2 target mRNAs of several host proteins involved in regulation of the cellular secretory apparatus (25). A recombinant virus that does not express these miRNAs did not induce formation of cVACs and produced significantly fewer infectious virions.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

Das

Ortiz

Gurczynski

et al. 2014

J Virol

View full text Add to dashboard Cite

Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

Das

Ortiz

Gurczynski

et al. 2014

J Virol

View full text Add to dashboard Cite

show abstract

“…47,48 Moreover, the Rho GTPase CDC42 is miRNA-regulated and plays a role in HCMV AC morphogenesis along with RAB5C, RAB11A and SNAP23. 49 Herein, we addressed the role of RhoB during human cytomegalovirus lytic infection. We extensively examined the subcellular distribution of RhoB both in fixed and live HCMVinfected cells and found that RhoB translocates to the cytoplasmic area that corresponds to the HCMV AC.…”

Section: Introductionmentioning

confidence: 99%

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

et al. 2015

View full text Add to dashboard Cite

Human Cytomegalovirus (HCMV), an ubiquitous b-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly a-and g-herpesviruses, whereas b-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection.

show abstract

“…It has been proposed that as nucleocapsids exit the nucleus, they traverse the layers of the cVAC where tegument acquisition and envelopment occur (1). Conditions that perturb cVAC morphology result in reduced production of infections virions (3)(4)(5)(6). Thus, although not essential for virus assembly and maturation, the cVAC is required for efficient production of infectious HCMV virions.…”

mentioning

confidence: 99%

Potent Inhibition of Human Cytomegalovirus by Modulation of Cellular SNARE Syntaxin 5

Cruz

Streck

Ferguson

et al. 2017

J Virol

View full text Add to dashboard Cite

Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient human cytomegalovirus (HCMV) assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. Although a recent screen identified three viral proteins essential for cVAC formation, less is known about the contribution of cellular factors. We show that HCMV infection increases the protein level of a cellular trafficking factor, syntaxin 5 (STX5), a member of the syntaxin family of SNARE proteins. STX5 is recruited to the cVAC in infected cells and is required for the efficient production of infectious virions. We find that STX5 is important for normal cVAC morphology and the proper localization of viral proteins. A previously identified inhibitor of trafficking, Retro94, causes the mislocalization of STX5, an altered cVAC morphology, and dispersal of viral proteins. The presence of Retro94 results in severely impaired production of infectious virions, with a decrease as great as 5 logs. We show that this inhibition is conserved among different strains of HCMV and the various cell types that support infection, as well as for murine CMV. Thus, our data identify a key cellular trafficking factor important for supporting HCMV infection.IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe disease and mortality in immunocompromised individuals, including organ transplant and AIDS patients. In addition, infection of a developing fetus may result in lifelong complications such as deafness and learning disabilities. Understanding in detail the processes involved in HCMV replication is important for developing novel treatments. One of these essential processes, assembly of infectious virions, takes places in the cytoplasmic viral assembly compartment. We identify a cellular protein, syntaxin 5, important for generating this compartment, and show that it is required for the efficient production of infectious virions. We also show that a small molecule that disrupts this protein also significantly reduces the amount of infectious virions that are generated. Thus, by pinpointing a cellular protein that is important in the replication cycle of HCMV, we identified a novel target that can be pursued for therapeutic intervention.

show abstract

Cytomegalovirus miRNAs Target Secretory Pathway Genes to Facilitate Formation of the Virion Assembly Compartment and Reduce Cytokine Secretion

Cited by 123 publications

References 45 publications

Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

Potent Inhibition of Human Cytomegalovirus by Modulation of Cellular SNARE Syntaxin 5

Contact Info

Product

Resources

About