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
. 2016 Sep 12;90(19):8621-33.
doi: 10.1128/JVI.00621-16. Print 2016 Oct 1.

MORC3, a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus

Elizabeth Sloan  1 , Anne Orr  2 , Roger D Everett  2
Affiliations

MORC3, a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus

Elizabeth Sloan et al. J Virol. .

Abstract

We previously reported that MORC3, a protein associated with promyelocytic leukemia nuclear bodies (PML NBs), is a target of herpes simplex virus 1 (HSV-1) ICP0-mediated degradation (E. Sloan, et al., PLoS Pathog 11:e1005059, 2015, http://dx.doi.org/10.1371/journal.ppat.1005059). Since it is well known that certain other components of the PML NB complex play an important role during an intrinsic immune response to HSV-1 and are also degraded or inactivated by ICP0, here we further investigate the role of MORC3 during HSV-1 infection. We demonstrate that MORC3 has antiviral activity during HSV-1 infection and that this antiviral role is counteracted by ICP0. In addition, MORC3's antiviral role extends to wild-type (wt) human cytomegalovirus (HCMV) infection, as its plaque-forming efficiency increased in MORC3-depleted cells. We found that MORC3 is recruited to sites associated with HSV-1 genomes after their entry into the nucleus of an infected cell, and in wt infections this is followed by its association with ICP0 foci prior to its degradation. The RING finger domain of ICP0 was required for degradation of MORC3, and we confirmed that no other HSV-1 protein is required for the loss of MORC3. We also found that MORC3 is required for fully efficient recruitment of PML, Sp100, hDaxx, and γH2AX to sites associated with HSV-1 genomes entering the host cell nucleus. This study further unravels the intricate ways in which HSV-1 has evolved to counteract the host immune response and reveals a novel function for MORC3 during the host intrinsic immune response.

Importance: Herpesviruses have devised ways to manipulate the host intrinsic immune response to promote their own survival and persistence within the human population. One way in which this is achieved is through degradation or functional inactivation of PML NB proteins, which are recruited to viral genomes in order to repress viral transcription. Because MORC3 associates with PML NBs in uninfected cells and is a target for HSV-1-mediated degradation, we investigated the role of MORC3 during HSV-1 infection. We found that MORC3 is also recruited to viral HSV-1 genomes, and importantly it contributes to the fully efficient recruitment of PML, hDaxx, Sp100, and γH2AX to these sites. Depletion of MORC3 resulted in an increase in ICP0-null HSV-1 and wt HCMV replication and plaque formation; therefore, this study reveals that MORC3 is an antiviral factor which plays an important role during HSV-1 and HCMV infection.

PubMed Disclaimer

Figures

FIG 1
FIG 1
MORC3 associates with Sp100 in PML NBs and ICP0 early during wt HSV-1 infection and is subsequently degraded. HFs were either mock infected or infected with wt HSV-1 (MOI of 2), fixed, and permeabilized at 1 or 2 h postinfection. Cells were analyzed for association of MORC3 (Novus Biologicals) (red) with Sp100 (top row, showing concentrations of MORC3 associated with Sp100 in a variable manner; see also Fig. 4) and with ICP0 (11060) (green) in infected cells using confocal microscopy. The blue signal in the merged channel is DAPI. The arrow indicates a cell with ICP0 in association with MORC3. The MORC3 signal diminishes as infection progresses.
FIG 2
FIG 2
MORC3 is degraded in an ICP0-dependent manner. HFT cells were mock infected (m) or infected at an MOI of 20 with an ICP0 null HSV-1 (ΔICP0) and lysed at 3, 6, and 9 h p.i. for Western blot analysis. Analysis of PML abundance and wt HSV-1-infected (MOI of 2) lysates prepared in parallel were included as positive controls for degradation. Membranes were probed with anti-MORC3 (Novus Biologicals) and anti-PML (5E10) antibodies, with anti-tubulin (Sigma-Aldrich) used as a loading control. Viral proteins ICP4 and UL42 were included to show equivalent infection between wt and ΔICP0 HSV-1.
FIG 3
FIG 3
RING finger domain of ICP0 is required for MORC3 degradation. (A) Schematic diagram of ICP0 displaying internal domains and SIM-like sequences (SLS) as well as regions of mutations/deletions of the RING finger (FXE) and C-terminal (E52X) domains. NLS, nuclear localization signal. (B) HA-TetR (control) and HA-cICP0, -cICP0 FXE (Δ149-160), -cICP0 E52X (Δ594-775), -cICP0 mSLS4, and -cICP0 mSLS457 cells with the ability to express wt and mutant forms of ICP0 were treated with doxycycline (Dox) (+) or left untreated (−) and then analyzed by Western blotting. Membranes were probed with anti-MORC3 (Novus Biologicals), anti-ICP0 (11060), and anti-PML (5E10) antibodies, with anti-tubulin (Sigma-Aldrich) included as a loading control.
FIG 4
FIG 4
MORC3 is recruited to HSV-1 genomes during the initial stage of infection. HFs were infected at low MOI with dl0Y4 for 24 h, fixed, permeabilized, and probed with anti-MORC3 antibody (Novus Biologicals) (purple). Anti-Sp100 (Sp26) (red) was included as a positive control for recruitment to viral genomes, with nuclei stained with DAPI (blue). Recruitment to ICP4 was visualized by confocal microscopy (right column) and compared to that of a typical uninfected cell (left column).
FIG 5
FIG 5
Characterization of MORC3-depleted cells. (A) Generation of MORC3-depleted HFT (HFT-shMORC3-1335 and -1339 cells) and HepaRG cells (HA-shMORC3-1335, -1337, and -1339 cells) using shRNAs expressed from lentiviral vectors. Total protein lysates were analyzed by Western blotting to determine the level of MORC3 depletion with HFT-shNeg and HepaRG cells included as controls. Tubulin was included as a loading control. (B and C) Characterization of MORC3-depleted HFT cells (B) and HepaRG cells (C). Mock HFT- and HA-shMORC3-1339 and control cells were immunostained for PML (red) and MORC3 (green) (left) as well as Sp100 (green) and PML (red) (right) to confirm MORC3 depletion and assess effects on PML NBs. DAPI staining of the nuclei is shown in blue in the merged panels.
FIG 6
FIG 6
Recruitment of PML NB proteins to viral DNA is diminished in the absence of MORC3. (A) HFT-shMORC3-1339 and HFT-shNeg cells were infected with dl0Y4 (MOI of 2) for 24 h. Cells were immunostained using PML (5E10) (red) and MORC3 (Novus Biologicals) (blue), and nuclei were stained with DAPI (far right) and visualized by confocal microscopy. Infected or presumed infected cells surrounding a plaque were counted and divided into three categories, which are represented here: category 1, ICP4+/PML+ (foci of ICP4 and PML in close association at the nuclear periphery of a cell); category 2, ICP4−/PML+ (foci of PML at the nuclear periphery of a cell in the pattern typical of an infected cell, but prior to detectable expression of ICP4); and category 3, ICP4+/PML− (ICP4 foci only at the nuclear periphery of a cell). (B) Percentages of cells within each category are presented in bar graphs. Sp100, hDaxx, and γH2AX were also assessed as described for PML, with results represented as bar graphs. (C) HFT-shMORC3-1339 and HFT-shNeg cells were infected with dl0Y4 (MOI of 2) for 24 h. Cells were immunostained using Sp100 (Sp26) (red) and PML (5E10) (blue) and nuclei were stained with DAPI (far right), while ICP4 was detected by the EYFP signal.
FIG 7
FIG 7
MORC3 has antiviral activity during ICP0-null mutant HSV-1 and wt HCMV infection. (A) Data from several independent viral plaque assays of both wt- and ΔICP0 HSV-1-infected HFT-shMORC3-1335, HFT-shMORC3-1339, HA-shMORC3-1335, and HA-shMORC3-1339 cells were averaged and normalized to the respective shNeg cell controls and plotted ± standard deviations. (B) Western blot analysis comparing infection efficiency of ΔICP0 HSV-1 within HFT-shMORC3-1339 and HFT-shNeg cells over a time course infection. Cells were infected at an MOI of 2 with ΔICP0 HSV-1, lysed at 2, 4, or 6 h p.i., and probed for ICP4, ICP0, UL42, and actin loading control. Mock-infected cells (m) were included as a control. (C) HCMV viral plaque-forming efficiencies using HFT-shMORC3-1335 and HFT-shMORC3-1339 cells normalized to HFT-shNeg cells and plotted ± standard deviations.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Knipe D, Howley P, Griffin D, Lamb R, Martin M, Roizman B, Strauss S. 2006. Fields Virology. Lippincott Williams and Wilkins, Philadelphia, PA.
    1. Weller S. 2011. Alphaherpesviruses. Caister Academic Press, Norfolk, United Kingdom.
    1. Boutell C, Everett RD. 2013. Regulation of alphaherpesvirus infections by the ICP0 family of proteins. J Gen Virol 94:465–481. doi:10.1099/vir.0.048900-0. - DOI - PubMed
    1. Geoffroy MC, Chelbi-Alix MK. 2011. Role of promyelocytic leukemia protein in host antiviral defense. J Interferon Cytokine Res 31:145–158. doi:10.1089/jir.2010.0111. - DOI - PubMed
    1. Everett RD, Boutell C, Hale BG. 2013. Interplay between viruses and host sumoylation pathways. Nat Rev Microbiol 11:400–411. doi:10.1038/nrmicro3015. - DOI - PubMed

Publication types

Substances

LinkOut - more resources