Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy

Schauflinger, Martin; Villinger, Clarissa; Mertens, Thomas; Walther, Paul; Einem, Jens von

doi:10.1111/cmi.12077

Cited by 50 publications

(85 citation statements)

References 47 publications

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“…The recent use of advanced electron microscopy techniques allows clear visualization of these steps in envelopment (64,65). Contact of the viral particle with a vesicle membrane initiates the formation of a crescent shape whereby the membrane wraps around the tegumented nucleocapsid.…”

Section: Discussionmentioning

confidence: 99%

Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types

Belzile

Sabalza

Craig

et al. 2016

J Virol

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Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem in immunocompromised individuals and has been associated with atherosclerosis. Previous studies have shown that the induction of autophagy can inhibit the replication of several different types of DNA and RNA viruses. The goal of the work presented here was to determine whether constitutive activation of autophagy would also block replication of HCMV. Most prior studies have used agents that induce autophagy via inhibition of the mTOR pathway. However, since HCMV infection alters the sensitivity of mTOR kinase-containing complexes to inhibitors, we sought an alternative method of inducing autophagy. We chose to use trehalose, a nontoxic naturally occurring disaccharide that is found in plants, insects, microorganisms, and invertebrates but not in mammals and that induces autophagy by an mTOR-independent mechanism. Given the many different cell targets of HCMV, we proceeded to determine whether trehalose would inhibit HCMV infection in human fibroblasts, aortic artery endothelial cells, and neural cells derived from human embryonic stem cells. We found that in all of these cell types, trehalose induces autophagy and inhibits HCMV gene expression and production of cell-free virus. Treatment of HCMV-infected neural cells with trehalose also inhibited production of cell-associated virus and partially blocked the reduction in neurite growth and cytomegaly. These results suggest that activation of autophagy by the natural sugar trehalose or other safe mTOR-independent agents might provide a novel therapeutic approach for treating HCMV disease. IMPORTANCEHCMV infects multiple cell types in vivo, establishes lifelong persistence in the host, and can cause serious health problems for fetuses and immunocompromised individuals. HCMV, like all other persistent pathogens, has to finely tune its interplay with the host cellular machinery to replicate efficiently and evade detection by the immune system. In this study, we investigated whether modulation of autophagy, a host pathway necessary for the recycling of nutrients and removal of protein aggregates, misfolded proteins, and pathogens, could be used to target HCMV. We found that autophagy could be significantly increased by treatment with the nontoxic, natural disaccharide trehalose. Importantly, trehalose had a profound inhibitory effect on viral gene expression and strongly impaired viral spread. These data constitute a proof-of-concept for the use of natural products targeting host pathways rather than the virus itself, thus reducing the risk of the development of resistance to treatment. H uman cytomegalovirus (HCMV) infects multiple cell types, including endothelial, smooth muscle, epithelial, and fibroblast cells, monocytes/macrophages, bone marrow progenitor cells, and cells of the neural lineage, and establishes lifelong persistence in the host (for a review, see reference 1). Seroprevalence is high in the general population, and in most healthy individuals, the in...

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Section: Discussionmentioning

confidence: 99%

Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types

Belzile

Sabalza

Craig

et al. 2016

J Virol

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“…This is followed by initial tegumentation of the capsid and transit of the encapsidated particles across the nuclear membrane (which includes primary envelopment and de-envelopment) (as reviewed in (Johnson and Baines, 2011; Mettenleiter et al, 2009)). Completion of tegumentation and re-envelopment of capsids occurs in the cytoplasm (Sanchez et al, 2000a; Sanchez et al, 2000b; Schauflinger et al, 2013). Viral reproduction concludes with these particles being shed in secretory vesicles via fusion with the plasma membrane (reviewed in reference Colberg-Poley and Williamson, 2013).…”

Section: Introductionmentioning

confidence: 99%

Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53

et al. 2016

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Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm.

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“…Nuclei take on a kidney-like (reniform) shape as they bend partially around the cVAC (5). Electron microscopic evidence indicates that the cVAC is the site of final tegumentation and envelope acquisition (6)(7)(8). Viral tegument proteins, envelope proteins, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments appear to radiate from a microtubule organizing center in the cVAC (4,(9)(10)(11).…”

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confidence: 99%

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

Das

Ortiz

Gurczynski

et al. 2014

J Virol

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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.

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Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy

Cited by 50 publications

References 47 publications

Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types

Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types

Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53

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

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