Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug 2;108(31):12869-74.
doi: 10.1073/pnas.1109796108. Epub 2011 Jul 18.

Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress

Sean T H Liu  1 , Ronit Sharon-Friling, Pavlina Ivanova, Stephen B Milne, David S Myers, Joshua D Rabinowitz, H Alex Brown, Thomas Shenk
Affiliations

Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress

Sean T H Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Human cytomegalovirus induces and requires fatty acid synthesis. This suggests an essential role for lipidome remodeling in viral replication. We used mass spectrometry to quantify glycerophospholipids in mock-infected and virus-infected fibroblasts, as well as in virions. Although the lipid composition of mock-infected and virus-infected fibroblasts was similar, virions were markedly different. The virion envelope contained twofold more phosphatidylethanolamines and threefold less phosphatidylserines than the host cell. This indicates that the virus buds from a membrane with a different lipid composition from the host cell as a whole. Compared with published datasets, the virion envelope showed the greatest similarity to the synaptic vesicle lipidome. Synaptosome-associated protein of 25 kDa (SNAP-25) is a component of the complex that mediates exocytosis of synaptic vesicles in neurons; and its homolog, SNAP-23, functions in exocytosis in many other cell types. Infection induced the relocation of SNAP-23 to the cytoplasmic viral assembly zone, and knockdown of SNAP-23 inhibited the production of virus. We propose that cytomegalovirus capsids acquire their envelope by budding into vesicles with a lipid composition similar to that of synaptic vesicles, which subsequently fuse with the plasma membrane to release virions from the cell.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Glycerophospholipids in mock-infected vs. HCMV-infected fibroblasts. (A) Total glycerophospholipid levels change only modestly during infection. Glycerophospholipids were measured by liquid chromatography-mass spectrometry (LC-MS) profiling of mock vs. infected cells at 0 (red), 6 (purple), 24 (yellow), 48 (green), 72 (blue), and 96 (white) hpi. Gray solid lines and dashed lines indicate a twofold and fivefold change in amount in infected vs. mock, respectively. Lipids with different head groups are shown by different symbols: PA (◆); PE (■); PG (▲); PI (×); PS (▬▬▬); and PC (+). The number of individual lipid species measured was PA, 20; PE, 37; PG, 24; PI, 17; PS, 23; and PC, 25. (B) PA (32:0) levels increase in primary fibroblasts with time after infection with HCMV. Data are from Dataset S1. Red triangles, infected cells; blue circles, mock-infected cells. *P < 0.05 and **P < 0.01, Student t test. Error bars indicate SEM.
Fig. 2.
Fig. 2.
Glycerophospholipids in virions compared with flbroblasts. (A) Modest differences between virions and mock-infected or HCMV-infected cells in terms of glycerophospholipid acyl chain length or degree of unsaturation. Acyl chain lengths were determined for lipids containing 30–42 total carbons in their two fatty acids combined. Percentages were calculated from Datasets S1 and S2. (B) HCMV virion and synaptic vesicle glycerophospholipids share similar head group composition. The percentage of each species in fibroblasts at 96 h after mock-infection or HCMV infection was calculated and compared with the virion lipidome. Synaptic vesicle lipidome data are from Takamori et al. (20).
Fig. 3.
Fig. 3.
SNAP-23 is relocalized to the assembly compartment after HCMV infection. (A) SNAP-23 protein levels remain nearly constant after infection. MRC5 fibroblasts were harvested at various times after infection at a multiplicity of 3 TCID50 units/mL, and SNAP-23 protein was assayed by Western blot. IE1, pUL99, and β-actin were monitored as controls. (B) SNAP-23 is colocalized with markers of the assembly compartment. MRC5 fibroblasts were analyzed by immunofluoresence at 96 hpi using antibodies to SNAP-23 (green) and pUL99 (red, upper infected panels) or pUL55 (red, lower infected panels).
Fig. 4.
Fig. 4.
SNAP-23 is required for the efficient production of infectious virus. (A) siRNA knockdown of SNAP-23. Twenty-four hours before infection, MRC5 fibroblasts received no treatment (No siRNA) or were transfected with a nontargeting (NT) siRNA or with an SNAP-23–specific siRNA. Cells were infected at a multiplicity of 3 TCID50 units/mL and harvested at 72 hpi. Lysates were prepared and assayed by Western blot using antibodies to the indicated proteins. Mock-infected cells that were not treated with an siRNA were assayed as a control. (B) siRNA knockdown of SNAP-23 impairs HCMV replication. Cells were pretreated for 24 h with siRNA to HCMV IE2 or SNAP-23, infected at a multiplicity of 0.5 TCID50 unit/mL, supernatants were harvested at 72 hpi, and virus was quantified by assaying for IE1 expression by immunofluorescence. Error bars indicate SEM. (C) shRNA knockdown of SNAP-23. Cells containing lentiviruses expressing SNAP-23–specific shRNAs (shRNA1-3) or a nontargeting shRNA (NT shRNA) were assayed by Western blot using antibodies to the indicated proteins. (D) shRNA knockdown of SNAP-23 impairs HCMV replication. Fibroblasts expressing shRNA1 or NT siRNA were infected at a multiplicity of 3 pfu per cell, and virus from the media of infected cultures was quantified by TCID50 assay at various times after infection. Error bars indicate SEM. (E) shRNA knockdown of SNAP-23 does not interfere with accumulation or localization of pUL99. Infected cells were monitored for expression of pUL99 by immunofluorescence (green), and nuclei were stained with DAPI (blue).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mocarski ES, Shenk T, Pass RF. Cytomegaloviruses. 5th Ed. Philadelphia: Lippincott Williams & Wilkins; 2007.
    1. Munger J, Bajad SU, Coller HA, Shenk T, Rabinowitz JD. Dynamics of the cellular metabolome during human cytomegalovirus infection. PLoS Pathog. 2006;2:e132. - PMC - PubMed
    1. Munger J, et al. Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotechnol. 2008;26:1179–1186. - PMC - PubMed
    1. AuCoin DP, Smith GB, Meiering CD, Mocarski ES. Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation. J Virol. 2006;80:8199–8210. - PMC - PubMed
    1. Das S, Vasanji A, Pellett PE. Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus. J Virol. 2007;81:11861–11869. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources