Cytomegalovirus Pathogenesis and Host Interactions

Editorial

07 July 2021

Editorial: Cytomegalovirus Pathogenesis and Host Interactions

Emma L. Poole

and

Michael M. Nevels

5,003 views

3 citations

Editors

Emma Poole

University of Cambridge

Michael M Nevels

University of St Andrews

Impact

(A) Results for GLG1, NEDD4 and CHST14, proteins selectively rescued by MG132 but not bortezomib. The left hand panels show data from the complete MG132/bortezomib screen and the right hand panels show the MG132 (10 µM)/Leupeptin (200 µM) screen (12 hpi) described previously (Nightingale et al., 2018). (B) (Left panel) Immunoblot showing differential effects of proteasome inhibitors MG132 (MG) and bortezomib (bort) on GLG1 protein during HCMV infection (MOI 5, 12 hpi). (Right panel) Quantitation of GLG1 relative to GAPDH (internal loading control).

Brief Research Report

27 January 2021

Rapid Degradation Pathways of Host Proteins During HCMV Infection Revealed by Quantitative Proteomics

Kai-Min Lin

, 3 more and

Michael P. Weekes

4,774 views

7 citations

RPE-1 or MRC-5 cells were infected with TB40/BAC4 HCMV3F at MOI 0.5 or MOI 5, respectively, and subjected to live-cell confocal microscopy acquiring images at 20 min intervals. (A) Fluorescence profiles of single cells for mNeonGreen-ie1/2, mTagBFP2-e1, and SCP-mCherry [curves were generated by smoothing raw data using the Savitzky-Golay algorithm (10-neighbor), n = 19]. (B) RNA-FISH was performed on RPE-1 cells infected with TB40/BAC4 wild type HCMV-infected cells at indicated times postinfection (0 h = not infected). Fluorescence intensity of the fluorescently labeled probe was integrated over whole cell bodies. Each dot represents one cell (n ≥ 20; error bars represent SD). (C) Averaged and normalized signals (mean: thick line; SD: dotted line) of fluorescence reporter signals in A (green: mNeonGreen-ie1/2; blue: mTagBFP2-e1; magenta: mCherry-SCP) are compared to averaged and normalized RNA-FISH signals from (B). Error bars and error lines represent SD.

Brief Research Report

08 January 2021

A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms

Ulfert Rand

, 2 more and

Luka Cicin-Sain

4,520 views

13 citations

Brief Research Report

30 September 2020

Evidence for Tethering of Human Cytomegalovirus Genomes to Host Chromosomes

Katrin Mauch-Mücke

, 2 more and

Michael M. Nevels

6,798 views

11 citations

Paradigms of HCMV latency and reactivation. (A) Silencing of the MIEP is required for HCMV latency, and it has long been thought that reactivation depends on de-repression of the MIEP for re-expression of IE genes. (B) Our recent work sheds new light on the control of MIE gene re-expression in THP-1 cell line and CD34+ primary cell models of latency and reactivation. Specifically, it defines alternative promoters within intron A of the MIE locus that give rise to full length IE1 and IE2 proteins. The intronic promoters must also be silenced for latency, similar to the MIEP. An additional post-transcriptional/translational regulation is likely involved, as low levels of iP2-derived transcripts are present during latency. Strikingly, reactivation stimuli in both models induce re-expression of IE1 and IE2 predominantly from the intronic promoters and to a much lesser extent, if at all, from the MIEP. The activation of the intronic promoters is regulated, at least in part, by the host transcription factor, FOXO3a, associated with hematopoietic differentiation. Other viral and cellular factors likely contribute to regulation of the MIE intronic promoters.

Review

17 September 2020

Control of Immediate Early Gene Expression for Human Cytomegalovirus Reactivation

Donna Collins-McMillen

, 2 more and

Felicia Goodrum

Human cytomegalovirus (HCMV) is a beta herpesvirus that persists for life in the majority of the world's population. The persistence of HCMV in the human population is due to the exquisite ability of herpesviruses to establish a latent infection that evades elimination by the host immune response. How the virus moves into and out of the latent state has been an intense area of research focus and debate. The prevailing paradigm is that the major immediate early promoter (MIEP), which drives robust expression of the major immediate early (MIE) transactivators, is epigenetically silenced during the establishment of latency, and must be reactivated for the virus to exit latency and re-enter productive replication. While it is clear that the MIEP is silenced by the association of repressive chromatin remodeling factors and histone marks, the mechanisms by which HCMV de-represses MIE gene expression for reactivation are less well understood. We have identified alternative promoter elements within the MIE locus that drive a second or delayed phase of MIE gene expression during productive infection. In the context of reactivation in THP-1 macrophages and primary CD34+ human progenitor cells, MIE transcripts are predominantly derived from initiation at these alternative promoters. Here we review the mechanisms by which alternative viral promoters might tailor the control of viral gene expression and the corresponding pattern of infection to specific cell types. Alternative promoter control of the HCMV MIE locus increases versatility in the system and allows the virus to tightly repress viral gene expression for latency but retain the ability to sense and respond to cell type-specific host cues for reactivation of replication.

8,583 views

19 citations

HCMV inhibits the autophagic process. (A,B) Representative images of HK-2 cells treated or not (control) with EBSS (to induce autophagy) or treated with EBSS + Spautin 1 (to block starvation-induced autophagy). HK-2 cells were also infected with HCMV at MOI 1 (and treated with EBSS for the 4 last h of infection or not). Cells were fixed after 2 days (A) or 7 days (B) and immunostained for LC3 (autophagosome marker, green) and IEA (viral protein, red). Nuclei were subsequently stained with DAPI. Scale bars represent 10 μm. Infected cells are marked with *. Quantification of LC3-positive dots per cell in each condition (lower panels). In HCMV + EBSS condition, cells expressing IEA (inf.) and neighboring non-infected cells (non-inf.) were analyzed separately. *p < 0.05 (ANOVA, with a Tukey contrast). (C,D) Immunoblot analysis of IEA and LC3-I and II in mock- or HCMV-infected HK-2 cells (2 and 7 days p.i.). Cells were treated when indicated for 4 h with EBSS and/or chloroquine (CQ). Actin was used as a loading control. A representative Western blot is depicted and LC3-II/Actin ratio is shown below the blot.

Original Research

15 September 2020

Human Cytomegalovirus Inhibits Autophagy of Renal Tubular Epithelial Cells and Promotes Cellular Enlargement

Ana C. López Giuliani

, 9 more and

Laura R. Delgui

3,529 views

4 citations

Comparison of Illumina-sequenced viral genomes. Viral DNA was purified from stocks of viruses mCMV-WT.BAC, mCMV-Δm06W, and mCMV-Δm06L, sequenced on Illumina MiSeq, and aligned to the mCMV WT reference sequence (RefSeq: NC_004065.1, INSDC: U68299.1). The Integrative Genomics Viewer was used for visualization. Gray areas indicate matches to the reference sequence, whereas colored spots indicate SNVs or INDELs. Regions with no successful alignment to the reference sequence are left white. Arrows represent the positions of the indicated ORFs. (A) Comparison of the viral genomes at 4.8–6.8 kbp. (B) Comparison of the viral genomes at 203–219 kbp.

Brief Research Report

26 August 2020

Positive Role of the MHC Class-I Antigen Presentation Regulator m04/gp34 of Murine Cytomegalovirus in Antiviral Protection by CD8 T Cells

Sara Becker

, 6 more and

Niels A. Lemmermann

3,353 views

8 citations

mCMV productively infects BMMC. (A) C57BL/6-derived BMMC were centrifugally infected with mCMV-egfp at an MOI of 4 and stimulated with 1 μM ionomycin for the cultivation period of 3–5 days. Representative images show infected eGFP+ (green) BMMC, indicating virus spread in the cultures over time. Bar marker: 50 μm. (B) Scheme explaining the principle of Cre-mediated recombination generating mCMV-rec-egfp that expresses green fluorescent eGFP [modified from Becker et al. (2015)]. (C) Quantitation of eGFP+ plaques in C57BL/6 MEF indicator monolayers on day 3 after infection with supernatants from ionomycin-conditioned C57BL/6 (B6) and Cre-transgenic Mcpt5-cre (B6-Mcpt5-cre) BMMC infected at an MOI of 4 with either mCMV-flox-egfp (open circles) or mCMV-rec-egfp (filled circles) and propagated for 5 days. Symbols represent plaque counts for independent BMMC cultures. Median values are indicated. Dotted line: dl, detection limit. (D) Representative eGFP+ plaques in MEF monolayers on day 3 after infection with supernatants from the BMMC cultures. Bar marker: 50 μm.

Brief Research Report

25 August 2020

The Anti-apoptotic Murine Cytomegalovirus Protein vMIA-m38.5 Induces Mast Cell Degranulation

Julia K. Schmiedeke

, 4 more and

Niels A. Lemmermann

2,670 views

4 citations

TLR-induced cytokine responses in blood cells from CMV-infected patients and blood donors. Blood cells were left unstimulated or stimulated with PGN EB (TLR2 ligand, 5 μg/mL), poly(A:U) (TLR3 ligand, 20 μg/mL), lipopolysaccharide (LPS, TLR4 ligand, 100 μg/mL), Imiquimod (TLR7 ligand, 20 μg/mL), Resiquimod-R848 (TLR 7/8 ligand, 10 μg/mL) or CpG oligonucleotide type A (CpG ODN2216, TLR9 ligand, 3μg/mL). After 24 h, supernatants were collected and analyzed for cytokine levels. White circles indicate healthy controls and black circles indicate CMV-infected patients. N =16 CMV-infected patients and n = 18 healthy controls were evaluated. Blue circles indicate values below or above the assay's detection limits. Red lines represent mean values. Data were analyzed using the non-parametric Mann–Whitney U test. *p ≤ 0.05.

Brief Research Report

07 August 2020

Genetic and Functional Characterization of Toll-Like Receptor Responses in Immunocompetent Patients With CMV Mononucleosis

Giada Frascaroli

, 12 more and

Stefania Varani

4,660 views

8 citations

Simply model for the differentiation of primary HCMV-infected monocytes. We have loosely grouped the process of differentiation into 3 phases. Phase (1) Viral glycoproteins interact with cellular receptors during cell entry, which activates a variety of signaling cascade that in turn promote the early stages of differentiation (<72 h after infection). Phase (2) The activated signaling is sustained and continues to induce monocyte differentiation into a unique M1/M2 macrophage (72 h ~ 2 weeks after infection). Phase (3) Differentiated macrophages show chronic activation and long-term survival (months to years) and show expression of a full cascade of viral genes and production of infectious virions (> 2 weeks after infection).

Review

31 July 2020

The Differentiation of Human Cytomegalovirus Infected-Monocytes Is Required for Viral Replication

Chan-Ki Min

, 4 more and

Andrew D. Yurochko

Viral dissemination is a key mechanism responsible for persistence and disease following human cytomegalovirus (HCMV) infection. Monocytes play a pivotal role in viral dissemination to organ tissue during primary infection and following reactivation from latency. For example, during primary infection, infected monocytes migrate into tissues and differentiate into macrophages, which then become a source of viral replication. In addition, because differentiated macrophages can survive for months to years, they provide a potential persistent infection source in various organ systems. We broadly note that there are three phases to infection and differentiation of HCMV-infected monocytes: (1) Virus enters and traffics to the nucleus through a virus receptor ligand engagement event that activates a unique signalsome that initiates the monocyte-to-macrophage differentiation process. (2) Following initial infection, HCMV undergoes a “quiescence-like state” in monocytes lasting for several weeks and promotes monocyte differentiation into macrophages. While, the initial event is triggered by the receptor-ligand engagement, the long-term cellular activation is maintained by chronic viral-mediated signaling events. (3) Once HCMV infected monocytes differentiate into macrophages, the expression of immediate early viral (IE) genes is detectable, followed by viral replication and long term infectious viral particles release. Herein, we review the detailed mechanisms of each phase during infection and differentiation into macrophages and discuss the biological significance of the differentiation of monocytes in the pathogenesis of HCMV.

9,788 views

33 citations

Review

29 July 2020

Interplay Between Calcium and AMPK Signaling in Human Cytomegalovirus Infection

Diana M. Dunn

and

Joshua Munger

Calcium and AMPK signaling during HCMV infection. Calcium signaling contributes to increased intracellular calcium release through HCMV encoded genes, UL146, US28, UL37, and US21, and through viral entry via epidermal growth factor receptor (EGFR). HCMV infection also induces the expression of CaMKK and AMPK, both of which are crucial for viral replication through multiple cellular signals. HCMV infection also impacts neural cell development which is associated with loss of proper calcium response. Inhibition of CaMKK by STO-609 inhibits HMCV viral replication. AMPK inhibition by Compound C, or activation by metformin or AICAR, also results in a decrease in HCMV viral replication.

Calcium signaling and the AMP-activated protein kinase (AMPK) signaling networks broadly regulate numerous aspects of cell biology. Human Cytomegalovirus (HCMV) infection has been found to actively manipulate the calcium-AMPK signaling axis to support infection. Many HCMV genes have been linked to modulating calcium signaling, and HCMV infection has been found to be reliant on calcium signaling and AMPK activation. Here, we focus on the cell biology of calcium and AMPK signaling and what is currently known about how HCMV modulates these pathways to support HCMV infection and potentially contribute to oncomodulation.

9,394 views

20 citations

Effect of Cullin depletion on HCMV-induced SAMHD1 loss. (A) Schematic of the experimental workflow. HF cells in six-well plates were transfected with siRNA twice during 24 h and then infected with HCMV (Towne) at an MOI of 5. (B) HF cells were transfected with control siRNA or siRNAs for CUL1, CUL2, CUL3, or CUL4, and their mRNA levels were measured by qRT-PCR. The relative SAMHD1 mRNA level is normalized with the level of β-actin. Results shown are mean values and standard errors of three independent experiments. Values of *P < 0.05 and **P < 0.01 are indicated. (C) siRNA-transfected HF cells were mock-infected (M) or infected with HCMV as described in (A). Total cell lysates were prepared at 24 and 48 h post infection and immunoblotted with antibodies for SAMHD1, p21CIP1, IE1/IE2, p52 (UL44), pp28 (UL99), and β-actin. (D) The relative SAMHD1 protein level normalized with that of β-actin is shown on the graph. Data are shown as mean values with standard errors from three independent assays. Values of *P < 0.05 are indicated.

Original Research

30 July 2020

Degradation of SAMHD1 Restriction Factor Through Cullin-Ring E3 Ligase Complexes During Human Cytomegalovirus Infection

Seokhwan Hyeon

, 3 more and

Jin-Hyun Ahn

4,056 views

13 citations

IL-6 secretion by RNA1.2 mutant-infected cells after NF-κB inhibition or TPRG1L knockdown. (A) HFFF2 cells were mock-infected or infected with WT, ΔRNA1.2 or ΔTATA at MOI = 5. At 72 h p.i., the cells were incubated with fresh medium lacking (NT) or containing IKKα-and-IKKβ inhibitor BMS-345541 (IKKi) for a further 6 h. The medium was harvested, and secreted IL-6 was analyzed by ELISA. (B) Immortalized HFT cells were transduced with lentiviruses encoding a non-targeting shRNA negative control (Neg) or a TPRG1L-targeting shRNA (KD). After the establishment of stably expressing cell lines, the cells were infected with WT, ΔRNA1.2 or ΔTATA at MOI = 5. The cells were incubated with fresh medium between 72 and 78 h p.i., which was then harvested and analyzed for secreted IL-6 by ELISA. RNA was also isolated at 78 h p.i. and analyzed by RT-qPCR in (C). (C) Expression of TPRG1L mRNA in knockdown samples calculated relative to that of Neg samples. Data from four independent experiments are shown in each of (A–C). The error bars denote standard error values, and significant differences are marked by asterisks (p < 0.003, paired t-test; p < 0.05, Wilcoxon matched-pairs signed rank test). (D) MCP-1 and CXCL1 transcript levels were monitored during infection of HFFF2 cells with WT, ΔRNA1.2 or ΔTATA at MOI = 5. At 72 h p.i., the cells were incubated with fresh medium lacking or containing TNF-α for an additional 6 h. RNA was then harvested and analyzed by RT-PCR. The data are representative of three experiments.

Original Research

22 July 2020

Human Cytomegalovirus Long Non-coding RNA1.2 Suppresses Extracellular Release of the Pro-inflammatory Cytokine IL-6 by Blocking NF-κB Activation

Betty Lau

, 8 more and

Andrew J. Davison

4,103 views

14 citations

BET bromodomain inhibitor (BRDi) effects on human herpesvirus (HHV) infection. Diagram summarizes the published effects of BET BRDi treatment on viral gene expression of both HHV latent (Left) and lytic (Right) infections. Briefly, BET BRDi treatment of cells causes release of BRD4 (thick gray arrow) from DNA, repressing host transcription, reactivation of EBV and cell growth. Dissociation of BRD4 (and BRD2) from virus genomes can both inhibit DNA replication (EBV) and reactivate KSHV, whilst release and redistribution of P-TEFb causes both reactivation and lytic augmentation of HSV. Effects of BET BRDi on HCMV are largely unknown. Elements of figure not to scale. IE, immediate early; mRNA, messenger RNA; vDNA, viral DNA.

Mini Review

02 July 2020

Bromodomain Inhibitors as Therapeutics for Herpesvirus-Related Disease: All BETs Are Off?

Ian J. Groves

, 1 more and

Mark R. Wills

4,922 views

13 citations

Analysis of longitudinal HCMV virus load and HCMV specific CD3+ T cell IFNγ responses of D+R– Kidney transplant patients. Two example D+R– kidney transplant patients with primary HCMV infection, T cell responses (spot forming units per 106 CD3+ T cells) were measured by IFNγ FluoroSpot (green triangles connected by a solid line) to HCMV peptide pools covering pp65 and UL144, IE1 and IE2, pp71 and US3, and gB, as well as a polyclonal T cell stimulation as a positive control (“POS”). Virus load (copies/ml blood) was measured by QNAT of HCMV DNA (pink hexagons connected by a dashed line). Cyan lines show the mean magnitude of response (±standard error) of CD3+ T cell IFNγ responses seen in healthy seropositive individuals in the same age decade as the transplant recipient for each peptide pool (Jackson et al., 2017b). (A) Patient 365 is an example of a D+R– patient with resolution of DNAemia following the emergence of detectable CD3+ IFNγ responses to four HCMV lytic peptide pools. (B) Patient 352, in contrast, had DNAemia which recurred several times, despite also developing detectable HCMV specific CD3+ T cell IFNγ responses which are comparable in frequency to those seen age-matched in healthy seropositives.

Original Research

26 June 2020

Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay

Charlotte J. Houldcroft

, 11 more and

Mark R. Wills

7,102 views

14 citations

Original Research

15 June 2020

Whole-Genome Approach to Assessing Human Cytomegalovirus Dynamics in Transplant Patients Undergoing Antiviral Therapy

Nicolás M. Suárez

, 14 more and

Andrew J. Davison

Human cytomegalovirus (HCMV) is the most frequent cause of opportunistic viral infection following transplantation. Viral factors of potential clinical importance include the selection of mutants resistant to antiviral drugs and the occurrence of infections involving multiple HCMV strains. These factors are typically addressed by analyzing relevant HCMV genes by PCR and Sanger sequencing, which involves independent assays of limited sensitivity. To assess the dynamics of viral populations with high sensitivity, we applied high-throughput sequencing coupled with HCMV-adapted target enrichment to samples collected longitudinally from 11 transplant recipients (solid organ, n = 9, and allogeneic hematopoietic stem cell, n = 2). Only the latter presented multiple-strain infections. Four cases presented resistance mutations (n = 6), two (A594V and L595S) at high (100%) and four (V715M, V781I, A809V, and T838A) at low (<25%) frequency. One allogeneic hematopoietic stem cell transplant recipient presented up to four resistance mutations, each at low frequency. The use of high-throughput sequencing to monitor mutations and strain composition in people at risk of HCMV disease is of potential value in helping clinicians implement the most appropriate therapy.

3,958 views

20 citations