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. 2022 Jan 26;14(2):246.
doi: 10.3390/v14020246.

A Viral Long Non-Coding RNA Protects against Cell Death during Human Cytomegalovirus Infection of CD14+ Monocytes

Marianne R Perera  1 , Kathryn L Roche  2 , Eain A Murphy  3 , John H Sinclair  1
Affiliations

A Viral Long Non-Coding RNA Protects against Cell Death during Human Cytomegalovirus Infection of CD14+ Monocytes

Marianne R Perera et al. Viruses. .

Abstract

Long non-coding RNA β2.7 is the most highly transcribed viral gene during latent human cytomegalovirus (HCMV) infection. However, as yet, no function has ever been ascribed to β2.7 during HCMV latency. Here we show that β2.7 protects against apoptosis induced by high levels of reactive oxygen species (ROS) in infected monocytes, which routinely support latent HCMV infection. Monocytes infected with a wild-type (WT) virus, but not virus deleted for the β2.7 gene (Δβ2.7), are protected against mitochondrial stress and subsequent apoptosis. Protected monocytes display lower levels of ROS and additionally, stress-induced death in the absence of β2.7 can be reversed by an antioxidant which reduces ROS levels. Furthermore, we show that infection with WT but not Δβ2.7 virus results in strong upregulation of a cellular antioxidant enzyme, superoxide dismutase 2 (SOD2) in CD14+ monocytes. These observations identify a role for the β2.7 viral transcript, the most abundantly expressed viral RNA during latency but for which no latency-associated function has ever been ascribed, and demonstrate a novel way in which HCMV protects infected monocytes from pro-death signals to optimise latent carriage.

Keywords: apoptosis; human cytomegalovirus; latency; long non-coding RNA; oxidative stress; reactive oxygen species.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TMRE measures mitochondrial membrane potential (MMP) in monocytes. (A) TMRE, a fluorescent, positively charged stain, accumulates in actively respiring mitochondria because the mitochondrial matrix is negatively charged. When the mitochondrial membrane potential is dissipated, TMRE disperses. (B) Primary CD14+ monocytes isolated from peripheral blood were left untreated or treated with 20 μM FCCP, an uncoupler, for 10 min. Cells were then TMRE and Hoechst stained and photographed on a fluorescence microscope. Graph in (C) shows mean % TMRE + ve cells from samples analysed in triplicate. Significance was determined by a two-tailed Student’s t-test. **** = p < 0.0001.
Figure 2
Figure 2
β2.7 protects against cadmium chloride-induced mitochondrial stress in HCMV infected CD14+ monocytes. (A,B) CD14+ monocytes were mock infected or infected with a WT or Δβ2.7 HCMV Toledo virus. At 14 d.p.i, half the cells were treated with 50 μM cadmium chloride for 24 h. All cells were then TMRE and Hoechst stained and photographed on a fluorescence microscope in (A). Graph in (B) shows mean % of TMRE + ve cells over three independent biological repeats. Error bars show standard deviation. Significant difference between mock and WT or WT and Δβ2.7 was determined using a one-way ANOVA with Tukey post hoc analysis. (C) CD14+ monocytes were infected with WT or Δβ2.7 HCMV TB40/E-SV40-GFP. 2 d.p.i, cells were treated with 50 μM CdCl2 for 24 h and then TMRE and Hoechst stained. GFP + ve cells with and without visible TMRE staining were enumerated. Graph shows the mean from 4 independent biological repeats from different blood donors. Statistical significance was determined by two-tailed paired t-test. ** = p <0.01. (D) Representative photos of CD14+ monocytes infected with WT or Δβ2.7 TB40/E-SV40-GFP that were treated with CdCl2 for 24 h at 4 d.p.i, and then TMRE and Hoechst stained. n.s. = not significant.
Figure 3
Figure 3
β2.7 protects against cadmium-induced apoptosis in infected monocytes. CD14+ monocytes were mock, WT or Δβ2.7 HCMV Toledo infected and at 6 d.p.i were treated with 50 μM CdCl2 for 24 h. Protein lysates from these cells were assessed for levels of cleaved caspase-3 and the loading control actin by immunoblot.
Figure 4
Figure 4
β2.7 protects against high ROS levels in infected monocytes. Primary CD14+ monocytes were isolated from peripheral blood and either mock infected or infected with WT or Δβ2.7 HCMV Toledo. 6 d.p.i, cells were untreated (A) or treated with 50 μM CdCl2 (B) for 24 h. Cells were then stained with Hoechst and the superoxide dye, MitoSOX, and imaged by fluorescence microscopy. (C) Quantification of MitoSOX fluorescence/cell for experiments described in A and B, where graph shows mean and standard deviation for three replicate experiments. Significance was determined by a two-way ANOVA with Tukey post hoc testing. ns = not significant, ** = p < 0.01, and **** = p < 0.0001.
Figure 5
Figure 5
Lowering ROS levels with an antioxidant rescues Δβ2.7 virus from cadmium-induced apoptosis. (A,B) Primary CD14+ monocytes were treated with 50 μM cadmium chloride and increasing concentrations of the antioxidants (A) N-acetyl cysteine (NAC) or (B) MitoTEMPO. Then, 24 h post treatment, cells were stained with TMRE and Hoechst and imaged by fluorescence microscopy. (C) CD14+ monocytes were mock infected or infected with WT or Δβ2.7 HCMV Toledo. Next, 6 d.p.i, cells were treated with 50 μM CdCl2 with or without the antioxidant NAC. Finally, 24 h post treatment, protein lysates were harvested and immunoblotted for cleaved caspase-3, cleaved PARP and the loading control actin.
Figure 6
Figure 6
WT, but not Δβ2.7 infection upregulates SOD2 and GPX-1 in infected monocytes and bystander cells. (A) Electrons leaking from Complex I or III in the electron transport chain may react with oxygen to form superoxide. Superoxide is converted to hydrogen peroxide by the mitochondrial enzyme, SOD2, which is subsequently processed to water and oxygen by e.g., GPX-1 or PRDX1. (B) CD14+ monocytes were mock infected (M) or infected with WT or Δβ2.7 HCMV Toledo. 7 d.p.i, protein lysates from cells were harvested and assessed for SOD2 and actin levels by immunoblot. Graph shows densitometry from 3 independent biological repeats normalised to actin levels. Significance difference between Mock and WT and WT and Δβ2.7 was determined by one-way ANOVA with Tukey post hoc testing. ns = not significant, * = p < 0.05, and ** = p < 0.01. (C) Proteins from CD14+ monocytes infected with mock, WT or Δβ2.7 Toledo were harvested at 1, 4, and 7 d.p.i and immunoblotted for SOD2 and the loading control actin. (D) As described in (B), but protein lysates were immunoblotted for GPX-1 and actin. * = p < 0.05. (E) Supernatant from CD14+ monocytes that were mock, WT or Δβ2.7 Toledo infected was harvested at 7 d.p.i and either transferred directly (‘+S/N from’) or first UV inactivated (‘+UV S/N from’) and then placed onto uninfected CD14+ monocytes. Then, 3 days after treatment with supernatants, protein was harvested and immunoblotted for SOD2 and actin.
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