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. 2016 Dec 16;91(1):e01637-16.
doi: 10.1128/JVI.01637-16. Print 2017 Jan 1.

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

Linda Cruz  1 , Nicholas T Streck  1 , Kevin Ferguson  1 , Trisha Desai  2 , Dhimant H Desai  2 , Shantu G Amin  2 , Nicholas J Buchkovich  3
Affiliations

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

Linda Cruz et al. J Virol. .

Abstract

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.

Keywords: Golgi apparatus; HCMV; SNARE; assembly; assembly compartment; cytomegalovirus; protein trafficking.

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Figures

FIG 1
FIG 1
HCMV regulation of Golgi apparatus SNAREs during virion production. (A) Schematic showing Qa SNAREs involved in Golgi apparatus-related trafficking. (B) Western blot analysis of Qa SNAREs from lysates harvested from uninfected cells (Mock) or cells infected with HCMV (AD169) (MOI of 3) for 24, 48, 72, and 96 hpi. Values below depict fold change from mock levels, calculated from three independent experiments. (C) Immunofluorescence analysis showing STX5 (green) and GM130 (red) in HCMV-infected cells at 96 hpi. (D) Immunofluorescence analysis showing STX5 (green) localization in uninfected (Mock) or HCMV-infected cells at 24, 48, 72, and 96 hpi relative to pp28 tagged with mCherry (red). Nuclei shown in panels C and D are labeled with DAPI (blue). Scale bar, 10 μm.
FIG 2
FIG 2
STX5 is required for efficient infection. (A) Schematic showing design of the inducible miRNA-expressing locus inserted between UL34a and TRS1 of the AD169 strain. (B) Western blot analysis of STX5 protein at 96 hpi with miSTX5 (MOI of 3) in the presence or absence of doxycycline (Dox). (C) Growth curve analysis of viral titers from miLUC or miSTX5 infections treated with or without doxycycline (10 μg/ml) at 3, 6, 9, 12, and 15 dpi (MOI of 0.05). Data shown are composites of three independent replicates. (D) Bright-field images of CPE associated with infections from the experiment shown in panel C at 9 dpi. (E) Western blot analysis of IE1 and pp28 protein at 96 hpi with miSTX5 (MOI of 3) in the presence or absence of doxycycline. (F) Immunofluorescence analysis of pp28 and expression of GFP at 96 hpi in miSTX5-infected cells (MOI of 3) either untreated or treated with doxycycline (10 μg/ml). Nuclei were labeled with DAPI (blue). Scale bar, 10 μm.
FIG 3
FIG 3
Retro94 blocks the efficient production of infectious HCMV virions. (A) Growth curve analysis of HCMV (AD169; MOI of 3) in untreated (wild type, WT), vehicle-treated (DMSO), or Retro94-treated cells. Samples were harvested at 24, 48, 72, 96, 120, and 144 hpi. Data shown are from one of three independent replicates. (B) Immunofluorescence detection of STX5 in uninfected (Mock) and HCMV-infected cells (96 hpi) in the presence of DMSO or Retro94. The far right panel features enhanced contrast of Retro94 samples for easier visualization of STX5 displacement. Nuclei are labeled with DAPI (blue). Scale bar, 10 μm. (C) Western blot analysis of STX5 protein in uninfected (Mock) and HCMV-infected cells at 24, 48, 72, and 96 hpi. Samples were treated with DMSO or Retro94 (10 μM) from 2 hpi. The mock sample was treated with Retro94 for 96 h. (D) Growth curve analysis of HCMV (MOI of 0.05) in DMSO- or Retro94-treated cells. Samples were harvested at 3, 6, 9, 12, and 15 dpi. Asterisks indicate the exchange of medium with fresh Retro94 at day 6. Data shown are from one of four independent replicates. (E) Bright-field images of CPE associated with infections from the experiment shown in panel C. (F) The viability of cells treated with DMSO or Retro94 for 4, 9, and 15 days. Fresh Retro94 was added at day 6. Bright-field images of cell monolayers at 4 days posttreatment are shown to the right of the graph. Brefeldin A was included as a positive control for cell death.
FIG 4
FIG 4
Retro94 alters morphology of the HCMV assembly compartment. (A) Western blot analysis of uninfected cells (Mock) or HCMV-infected cells (AD169; MOI of 3) treated with DMSO or Retro94 (10 μM) at 24, 48, 72, and 96 hpi. (B) DNA replication analysis of cells infected at an MOI of 3 and harvested at 24, 48, 72, and 96 hpi. Samples were treated with DMSO, Retro94 (10 μM), or acyclovir (100 μg/ml). Data are composites of three independent replicates. (C) Localization of viral proteins in HCMV-infected cells at 96 hpi in the presence of DMSO or Retro 94 (10 μM). pp28 was detected by tagging with mCherry, and gB was detected by immunofluorescence. (D) Immunofluorescence detection of STX5 (green) in HCMV-infected cells (MOI of 3) at 96 hpi treated with DMSO or Retro94. pp28-mCherry is shown in red for reference. (E) Virus titers at 96 hpi of HCMV-infected cells treated with Retro94 at 2, 24, 48, or 72 hpi. *, P < 0.05; N.S., not significant. (F) Localization of pp28-mCherry in samples corresponding to those shown in panel E. (G) Immunofluorescence detection of pp28 in HCMV-infected cells subjected to either treatment with Retro94 throughout infection or treatment with Retro94 and subsequent withdrawal of Retro94 at 9 dpi followed by recovery for an additional 9 days. Nuclei shown in panels C, D, F, and G are labeled with DAPI (blue). Scale bar, 10 μm.
FIG 5
FIG 5
Retro94 results in reduced cytoplasmic viral activity. Electron micrographs of HCMV-infected cells (AD169; MOI of 3) at 96 hpi treated with DMSO (A) or Retro94 (B to F) at 2 hpi. Asterisks in panels B to D indicate areas of juxtanuclear accumulation of membranes. (E and F) High-magnification images of large, dense, membrane-enwrapped structures found at the periphery of areas of juxtanuclear membrane accumulation. AC, assembly compartment. Nuc, nucleus.
FIG 6
FIG 6
Juxtanuclear accumulation of Golgi fragments in the absence of STX5. (A) Immunofluorescence of Golgi complex (GM130) and TGN (p230) markers (green) in HCMV-infected cells (AD169; MOI of 3, 96 hpi) treated with DMSO or Retro94. Lower insets show single cells enlarged from the images above. A merged image with pp28-mCherry (red) is shown for reference. (B) Immunofluorescence of Golgi apparatus markers p115 (green) and pp28 (red) in cells infected with miSTX5 in the presence or absence of doxycycline at 96 hpi. (C) Proposed model for the role of STX5 during HCMV infection. Nuclei shown in panels A and B are labeled with DAPI (blue). Scale bar, 10 μm.
FIG 7
FIG 7
The Retro94-dependent inhibition of HCMV is not strain, cell type, or species specific. (A) Viral titers at 15 dpi of fibroblasts infected with various strains of HCMV and treated with DMSO or Retro94 at 2 hpi. The medium was changed, and fresh Retro94 was added at 6 dpi. Bright-field images representing CPE of infections are shown on the right. (B) HCMV-infected ARPE-19 cells treated with DMSO or Retro94 (10 μM). Infected cells were detected by monitoring mCherry (red) expressed by TB40 virus. Scale bar, 1 mm. The table below the images shows the number of foci detected, average area, and standard error of the mean (SEM). (C) Immunofluorescence analysis of STX5 (green) and pp28 (red) in HCMV-infected ARPE-19 cells at 96 hpi in the presence of DMSO or Retro94 (10 μM). (D) Viral titers at 6 dpi of MCMV-infected fibroblasts treated with DMSO or Retro94. (E) Immunofluorescence analysis of STX5 (green) and GM130 (red) in MCMV-infected NIH 3T3 cells at 72 hpi in the presence of DMSO or Retro94 (10 μM). (F) Growth curve analysis of HCMV (MOI of 3) in the DMSO control (No drug) or in cells treated with Retro94 and F-Retro94 at increasing concentrations. Samples were harvested at 96 hpi. Representative data shown are from one of four (Retro94) or one of three (F-Retro94) independent replicates. Values in A and D are composites of three independent experiments.
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