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. 2018 Feb 26;92(6):e01849-17.
doi: 10.1128/JVI.01849-17. Print 2018 Mar 15.

A Role for Myosin Va in Human Cytomegalovirus Nuclear Egress

Adrian R Wilkie  1 , Mayuri Sharma  1 , Jean M Pesola  1 , Maria Ericsson  2 , Rosio Fernandez  1 , Donald M Coen  3
Affiliations

A Role for Myosin Va in Human Cytomegalovirus Nuclear Egress

Adrian R Wilkie et al. J Virol. .

Abstract

Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the betaherpesvirus human cytomegalovirus (HCMV) and is important for nuclear egress and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. In this study, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and coimmunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress.IMPORTANCE Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of alphaherpesvirus capsids, a functional role for any specific myosin in nuclear egress has not been reported. Furthermore, the notion that an actomyosin-based mechanism facilitates intranuclear capsid movement is controversial. Here we show that human cytomegalovirus capsids associate with nuclear myosin Va and F-actin and that antagonism of myosin Va impairs capsid localization toward the nuclear rim and nuclear egress. Together with our previous results showing that nuclear F-actin is induced upon HCMV infection and is also important for these processes, our results lend support to the hypothesis that nascent human cytomegalovirus capsids migrate to the nuclear periphery via actomyosin-based movement. These results shed light on a poorly understood viral process and the cellular machinery involved.

Keywords: actin; cytomegalovirus; intranuclear movement; myosin; nuclear egress; replication compartment.

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Figures

FIG 1
FIG 1
HFFs were either mock infected or infected with WT HCMV (MOI of 1). At 72 hpi, nuclear and cytoplasmic fractions were prepared and then analyzed by Western blotting to assess myosin Va (MyoVa) expression levels, with tubulin and lamin B serving as fractionation controls for the cytoplasm and nucleus, respectively.
FIG 2
FIG 2
(A to C) HFFs were mock infected or infected with HCMV 44-F (MOI of 1) and fixed at 48 or 72 hpi. Coverslips were then stained with DAPI (blue), anti-FLAG (blue), anti-MCP (green), and anti-MyoVa (red) antibodies and imaged with spinning-disk confocal microscopy. In panels B and C, DAPI is excluded from the merged images. In panel B, arrows indicate MyoVa in RCs prior to detectable MCP expression. In panel C, white color indicates colocalization between UL44, MCP, and MyoVa. All images are single optical sections. Scale bars are 10 mm. (D) HFFs were infected with HCMV (MOI of 1). At 72 hpi, nuclear lysates were prepared and immunoprecipitated with resin conjugated to either an anti-Myosin Va or an IgG isotype control antibody. Lysates and eluates were then analyzed by Western blotting using antibodies against the proteins indicated at the right.
FIG 3
FIG 3
(A and B) HFFs were infected with HCMV (MOI of 1) and fixed for immuno-EM at 72 hpi. The cells were further processed by primary staining with anti-MyoVa or anti-IE 1/2 (negative control) antibodies, followed by secondary staining with 10-nm protein A-gold. Imaging was conducted using a transmission electron microscope. In the rightmost image in panel A, the white arrowheads indicate capsids (without DNA) associated with MyoVa and the black arrowhead indicates a capsid (without DNA) that is not associated with MyoVa. In the rightmost image in panel B, the black arrowheads indicate capsids that are not associated with IE 1/2 (leftmost capsids contain DNA; rightmost capsid does not contain DNA). Scale bars are 100 nm. (C) The percentage of capsids associated with at least one gold particle was calculated for each condition (MyoVa, n = 95; IE 1/2, n = 127). The P value was calculated using Fisher's exact test. ****, P < 0.0001.
FIG 4
FIG 4
(A) HFFs stably expressing LifeAct-GFP-NLS (green) were either mock infected (left) or infected with WT HCMV (MOI = 1) (right). At 72 hpi, cells were fixed, stained with anti-MCP (shown as blue [imaged in far red]) and anti-MyoVa (red) antibodies and DAPI (blue), and imaged with spinning-disk confocal microscopy. White color indicates colocalization between MCP, actin, and MyoVa. Scale bar is 10 mm. (B) To measure colocalization, the fluorescence intensity of each channel was plotted across the white line shown in the merged image. Images are single optical sections.
FIG 5
FIG 5
HFFs were transfected with a pool of MyoVa siRNA or NT siRNA (control), followed by infection with WT HCMV (MOI = 1). (A) Whole-cell lysates were harvested at 72 hpi and analyzed by Western blotting. Undiluted (neat) NT siRNA lysates were diluted as shown to quantify MyoVa knockdown. The numbers under the top portion represent band intensities relative to the neat lysates. (B) At 72 or 96 hpi, media were removed for titration to measure the production of infectious virus. The P values were calculated using an unpaired t test (3 independent experiments). ****, P < 0.0001; *, P = 0.045. (C) Cell monolayers were fixed and processed for EM. Capsids were counted in the nucleus and cytoplasm of whole-cell sections of 10 (72 hpi) or 7 (96 hpi) cells for each condition, with each point representing the number in a compartment in an individual cell section. Bars and the numbers alongside them indicate mean numbers of capsids. P values were calculated using the Mann-Whitney test. ns, not significant. P values were as follows: Nucleus, 72 hpi, P = 0.31; 96 hpi, P > 0.99; Cytoplasm, *, P = 0.04 (72 hpi) and P = 0.01 (96 hpi). (D) Cytoplasmic capsids as a percentage of total capsids (cytoplasmic and nuclear) for each cell. The P values were calculated using an unpaired t test. *, P = 0.02 (72 hpi) and P = 0.04 (96 hpi). (E) Lysates derived from NT or MyoVa siRNA-transfected cells were analyzed by Western blotting to assess late viral gene expression using antibodies against proteins indicated on the right.
FIG 6
FIG 6
(A) HFFs were transfected with NT siRNA, the pool of myosin Va siRNA, or each of the siRNAs that comprise the pool and then infected with WT HCMV (MOI = 1). (A) Whole-cell lysates were harvested at 72 hpi and analyzed by Western blotting to assess myosin Va knockdown. Also at 72 hpi, media were removed for titration to measure virus production (one experiment) (B), and cell monolayers were fixed and processed for EM. Capsids were counted in the nucleus and cytoplasm of whole-cell sections in 10 cells for each condition (C). P values were calculated using the Mann-Whitney test. ns, not significant (P = 0.31). *, P = 0.04. (D) Cytoplasmic capsids as a percentage of total capsids for each cell. The P value was calculated using an unpaired t test. *, P = 0.04.
FIG 7
FIG 7
Lentiviruses expressing LT-GFP-NLS or GFP-NLS (control) were generated and used to transduce HFFs. Doxycycline (Dox) was added to media to induce DN expression. Cells were harvested to assess DN mutant expression levels with Western blot analysis using antibodies against GFP (top) or actin as a loading control (bottom) (A) or fixed, stained with DAPI (blue), and imaged with spinning-disk confocal microscopy to assess subcellular localization of LT-GFP-NLS or GFP-NLS relative to DAPI (B). (C) LT-GFP-NLS- and GFP-NLS-transfected HFFs were infected with WT HCMV (MOI = 1) and Dox was added to media at 24 hpi to induce DN mutant expression. At 72 hpi, media were removed and titrated to measure the production of infectious virus. The P value was calculated using an unpaired t test (4 independent experiments). **, P = 0.007. (D) Cell monolayers were fixed and processed for EM to assess nuclear egress. Capsids were counted in the nucleus and cytoplasm of whole-cell sections in 12 cells for each condition. P values were calculated using the Mann-Whitney test. ns, not significant (P = 0.183); **, P = 0.003. (E) Cytoplasmic capsids as a percentage of total capsids (cytoplasmic and nuclear) for each cell. The P value was calculated using an unpaired t test. *, P = 0.02.
FIG 8
FIG 8
(A) LT-GFP-NLS- or GFP-NLS-expressing HFFs were infected with WT HCMV (MOI = 1). At 24 hpi, doxycycline was added to media to induce DN expression, and at 72 hpi cell monolayers were fixed for EM analysis. Capsids associated or not associated with electron-dense RC-like inclusions were counted in representative nuclear sections of 16 cells for each condition. White arrows indicate capsids located within RC-like inclusions; black arrows indicate capsids located outside RC-like inclusions. Nuc., nuclear compartment; Cyt., cytoplasmic compartment. (B) The percentage of capsids not associated with RC-like inclusions was calculated in each nucleus and plotted. (C) HFFs were transfected with siRNA 1 or 4 and then infected with WT HCMV (MOI = 1). At 72 hpi, cell monolayers were fixed for EM analysis. Capsids were counted in the nuclei of 10 cells for each condition and plotted. All P values were calculated using the Mann-Whitney test. ****, P < 0.0001; **, P = 0.003.
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