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. 2023 Aug 31;14(4):e0074923.
doi: 10.1128/mbio.00749-23. Epub 2023 Jul 21.

The US21 viroporin of human cytomegalovirus stimulates cell migration and adhesion

Anna Luganini  1 , Valentina Serra  1 , Giorgia Scarpellino  1 , Shree Madhu Bhat  1 , Luca Munaron  1 , Alessandra Fiorio Pla  1 , Giorgio Gribaudo  1
Affiliations

The US21 viroporin of human cytomegalovirus stimulates cell migration and adhesion

Anna Luganini et al. mBio. .

Abstract

The human cytomegalovirus (HCMV) US12 gene family contributes to virus-host interactions by regulating the virus' cell tropism and its evasion of host innate immune responses. US21, one of the 10 US12 genes (US12-US21), is a descendant of a captured cellular transmembrane BAX inhibitor motif-containing gene. It encodes a 7TMD endoplasmic reticulum (ER)-resident viroporin (pUS21) capable of reducing the Ca2+ content of ER stores, which, in turn, protects cells against apoptosis. Since regulation of Ca2+ homeostasis affects a broad range of cellular responses, including cell motility, we investigated whether pUS21 might also interfere with this cytobiological consequence of Ca2+ signaling. Indeed, deletion of the US21 gene impaired the ability of HCMV-infected cells to migrate, whereas expression of US21 protein stimulated cell migration and adhesion, as well as focal adhesion (FA) dynamics, in a way that depended on its ability to manipulate ER Ca2+ content. Mechanistic studies revealed pUS21-mediated cell migration to involve calpain 2 activation since its inhibition prevented the viroporin's effects on cell motility. Pertinently, pUS21 expression stimulated a store-operated Ca2+ entry (SOCE) mechanism that may determine the activation of calpain 2 by promoting Ca2+ entry. Furthermore, pUS21 was observed to interact with talin-1, a calpain 2 substrate, and crucial protein component of FA complexes. A functional consequence of this interaction was confirmed by talin-1 knockdown, which abrogated the pUS21-mediated increase in cell migration. Together, these results indicate the US21-encoded viroporin to be a viral regulator of cell adhesion and migration in the context of HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic pathogen that owes part of its success to the capture, duplication, and tuning of cellular genes to generate modern viral proteins which promote infection and persistence in the host by interfering with many cell biochemical and physiological pathways. The US21 viral protein provides an example of this evolutionary strategy: it is a cellular-derived calcium channel that manipulates intracellular calcium homeostasis to confer edges to HCMV replication. Here, we report on the characterization of a novel function of the US21 protein as a viral regulator of cell migration and adhesion through mechanisms involving its calcium channel activity. Characterization of HCMV multifunctional regulatory proteins, like US21, supports the better understanding of viral pathogenesis and may open avenues for the design of new antiviral strategies that exploit their functions.

Keywords: Ca2+ homeostasis; US21 protein; cell migration; human cytomegalovirus; viroporin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
US21 is required for the migration of HCMV-infected cells. Serum-starved HFFs were mock-infected or infected with TRwt, TRUS21-HA, or TRΔUS21 (MOI of 1 PFU/cell) and then analyzed in chemotactic migration assays. Data shown are means ± SEM of three independent experiments performed in triplicate and analyzed using unpaired t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs calibrator sample (mock).
Fig 2
Fig 2
pUS21 expression stimulates cell migration. (A) Tetracycline-inducible expression of pUS21-HA, pUS21-HA D178N, and pUS21-HA D201N in T-REx-U2OS-derived cell lines as verified by immunoblotting with an anti-HA MAb. Protein extracts were from cells non-induced (NI) or induced (I) with tetracycline 1 µg/mL for 24 and 48 h. (B) Expression of pUS21 lowers the agonist-releasable Ca2+ content in U2OS cells. Box plot 5th–95th percentiles show the maximum cytosolic Ca2+ concentrations in ionomycin/thapsigargin-stimulated Fura-2 AM-loaded T-REx-U2OS cell lines. Data were analyzed by one-way ANOVA followed by the Dunn’s multiple comparison test: **P < 0.01, ****P < 0.0001; pUS21-HA NI, n = 380 cells; pUS21-HA I, n = 425 cells; pUS21-HA D178N NI, n = 93 cells; pUS21-HA D178N I, n = 87 cells; pUS21-HA D201N NI, n = 116 cells; and pUS21-HA D201N I, n = 140 cells. (C) Random migration of T-REx-U2OS cells induced to express pUS21-HA, pUS21-HA D178N, or pUS21-HA D201N. Data are shown as box plot 5th–95th percentiles of three independent experiments performed in triplicate. Statistical significance vs NI cells: ****P < 0.0001; pUS21-HA NI, n = 84 cells; pUS21-HA I, n = 115 cells; pUS21-HA D178N NI, n = 96 cells; pUS21-HA D178N I, n = 105 cells; pUS21-HA D201N NI, n = 118 cells; and pUS21-HA D201N I, n = 102 cells.
Fig 3
Fig 3
pUS21 expression enhances cell adhesion. (A) Adhesion of T-REx-U2OS cells that express pUS21-HA, pUS21-HA D178N, or pUS21-HA D201N. T-REx-U2OS cells were left non-induced (NI) or induced (I) with 1 µg/mL tetracycline for 48 h to express pUS21-HA, pUS21-HA D178N, or pUS21-HA D201N proteins. Thereafter, cells were detached, counted, plated, and allowed to adhere for 40 min at 37°C. Adherent cells were then stained with DAPI and microscopically counted. Results are representative of three independent experiments performed in triplicate and showed as box plot 5th–95th percentiles. Statistical significance vs NI cells: ****P < 0.0001. For all conditions, the number of microscope fields analyzed for cell counting was n = 32, with an average number of cells/field of pUS21-HA NI, n = 59 cells/field; pUS21-HA I, n = 123 cells/field; pUS21-HA D178N NI, n = 53 cells/field; pUS21-HA D178N I, n = 115 cells/field; pUS21-HA D201N NI, n = 47 cells/field; and pUS21-HA D201N I, n = 47 cells/field. (B) pUS21 expression increases the number of focal adhesions (FAs). (Upper panel) Non-induced (NI) or induced (I) for 48 h T-REx-U2OS-US21-HA cells were fixed, permeabilized, and stained for paxillin. Representative images are shown. Figures in the bottom panels present enlargements of the inset (gray box) for both NI and I representative cell images. The number of FAs was determined by counting paxillin spots on the overall cell area (lower left panel) or at the cell perimeters (cortical) only (lower right panel) of 50 cells. Data were normalized according to cell area and are shown as box plots of 5th–95th percentiles. The average of FA/area for total counts (lower left panel) for pUS21-HA NI = 0.039 ± 0.018 and for pUS21-HA I = 0.079 ± 0.024; the average of FA/area for cortical counts (lower right panel) for pUS21-HA NI = 0.022 ± 0.009 and for pUS21-HA I = 0.043 ± 0.015. Examples of cortical FAs are indicated by white arrows; yellow arrows indicate intracellular FAs. Scale bars: 20 µM. Magnification: 60×. Statistical analysis vs NI cells: ****P < 0.0001.
Fig 4
Fig 4
The pUS21-mediated increase in cell migration involves calpain and SOCE activation. (A and B) Inhibition of calpain activity abrogates pUS21-induced cell migration. T-REx-U2OS cell lines, non-induced (NI) or induced (I) for 30 h, were treated with 50 µM ALLN (calpain 1/2 inhibitor) (A) or inhibitor IV (calpain 2 inhibitor) (B) from 4 h before and during time-lapse image acquisition for random migration analysis. Images were acquired at 10 min intervals for 10 h. Box plots of 5th–95th percentiles are shown. Statistical analysis vs NI cells: ****P < 0.0001. Panel A for untreated cells: pUS21-HA NI, n = 140 cells; pUS21-HA I, n = 124 cells; pUS21-HA D178N NI, n = 92 cells; pUS21-HA D178N I, n = 138 cells; pUS21-HA D201N NI, n = 132 cells; pUS21-HA D201N I, n = 108 cells. For cells treated with ALLN: pUS21-HA NI, n = 98 cells; pUS21-HA I, n = 94 cells; pUS21-HA D178N NI, n = 70 cells; pUS21-HA D178N I, n = 76 cells; pUS21-HA D201N NI, n = 109 cells; pUS21-HA D201N, I n = 85 cells. Panel B for untreated cells: pUS21-HA NI, n = 116 cells (left) and n = 97 cells (right); pUS21-HA I, n = 129 cells (left) and n = 128 cells (right); for cells treated with ALLN: pUS21-HA NI, n = 131 cells and pUS21-HA I, n = 110 cells; for cells treated with inhibitor IV: pUS21-HA NI, n = 75 cells and pUS21-HA I, n = 82 cells. (C) pUS21 expression stimulates calpain activity. T-REx-U2OS-US21-HA, T-REx-U2OS-US21-HA D178N, and T-REx-U2OS-US21-HA D210N cell lines were non-induced (NI) or induced (I) with 1 µg/mL tetracycline for 48 h. Then, 200 µg of cell protein extracts was incubated with the calpain substrate (Ac-LLY-AFC) for 1 h at 37°C. Calpain enzymatic assays were determined in four independent experiments performed in triplicate. Results are expressed as means ± SEM. Statistical analysis vs NI cells: ***P < 0.001, ****P < 0.0001. (D) SOCE activation contributes to pUS21-stimulated cell migration. T-REx-U2OS-US21-HA cells were left non-induced (NI) or induced (I) with tetracycline for 30 h. Immediately before time-lapse image acquisition, cells were treated with 20 µM BTP2. Images were acquired at 10 min intervals for 10 h using a Nikon Plan 20× objective and a CCD camera. Data are shown as box plots of 5th–95th percentiles. Statistical analysis vs NI cells: ****P < 0.0001. For untreated cells: pUS21-HA NI, n = 323 cells and pUS21-HA I, n = 334 cells; for cells treated with BTP2: pUS21-HA NI, n = 304 cells and pUS21-HA I, n = 343 cells.
Fig 5
Fig 5
The interaction of pUS21 with talin-1 is required for the pUS21-dependent stimulation of cell migration. (A) Interaction of pUS21 and talin-1 in T-REx-U2OS-US21-HA cells. Cell protein extracts were prepared from cells non-induced (NI) or induced (I) for 48 h and immunoprecipitated using anti-talin-1 MAb or a non-specific control mouse IgG. Then, immunoprecipitates and input cell extracts were analyzed by immunoblotting to detect talin-1 and HA epitopes. (B) pUS21 and talin-1 colocalize in infected HFFs. HFFs were infected with TRUS21-HA (left), TRΔUS21 (right) (MOI of 1 PFU/cell), or mock infected. At various times p.i., cells were fixed, permeabilized, and immunostained with anti-HA (green) and an anti-talin-1 (red) MAbs for TRUS21-HA-infected cells or with an anti-talin-1 (red) MAb and anti-IE2 (green) PAb for TRΔUS21-infected HFFs. The location of nuclei is indicated by N. Magnification: 63×. (C) Silencing of talin-1 protein expression by siRNAs. T-REx-U2OS-US21-HA cells were transiently transfected with 100 nM of 27-nucleotide oligo duplexes human talin-1 specific sequence (siRNA-1 and siRNA-2), or a scrambled siRNA control (a negative control for specific gene downregulation), or not transfected (CTRL). After 96 h, total cell extracts were prepared and analyzed by immunoblotting with mouse anti-talin-1 MAb and vinculin (control for protein loading). (D) Talin-1 expression is required for pUS21-mediated increase in cell migration. T-REx-U2OS-US21-HA cells were transiently transfected with 100 nM of human talin-1 siRNA-2 or scrambled siRNA. After 48 h, cells were left non-induced (NI) or induced (I) with tetracycline for 30 h before time-lapse image acquisition for random migration analysis. Images were acquired at 10 min intervals for 10 h. Data are displayed as box plots of 5th–95th percentiles for three independent experiments performed in triplicate. *P < 0.05, ****P < 0.0001. For non-transfected cells: pUS21-HA NI, n = 93 cells and pUS21-HA I, n = 115 cells; for cells transfected with talin-1 siRNA-2: pUS21-HA NI, n = 46 cells and pUS21-HA I, n = 54 cells; for cells transfected with scramble siRNA: pUS21-HA NI, n = 92 cells and pUS21-HA I, n = 87 cells.
Fig 6
Fig 6
Control of cell adhesion and migration by the US21 viroporin of HCMV. The channel function of pUS21 leads to depletion of ER Ca2+ content, which is then detected by the Stim1 sensor that, in turn, triggers the activation of SOCE and the influx of Ca2+ from the extracellular environment through SOC channels. The resulting increase in cytoplasmic Ca2+ activates calpain-2, talin-1 proteolysis, and the turnover of focal adhesions. Overall, this pUS21-dependent mechanism stimulates cell spread and increases the speed of cell migration. FA, focal adhesion; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase pump; SOC, store-operated Ca2+ channel; SOCE, store-operated Ca2+ entry mechanism; Stim1, stromal interaction molecule 1 that functions as an ER Ca2+ sensor.
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