Strategy of Human Cytomegalovirus To Escape Interferon Beta-Induced APOBEC3G Editing Activity

ABSTRACTThe apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) gene products and, to a lesser degree, those of A3F but not of A3A, are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon beta (IFN-β), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-β by limiting the presence of A3G hot spots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections.IMPORTANCEAPOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study, we show that particularly the APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and that its upregulation is mediated by IFN-β. Furthermore, we were able to demonstrate that neither A3G knockout nor A3G overexpression appears to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hot spots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteracting mechanisms under A3G-mediated selective pressure.

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The Zinc Finger Antiviral Protein ZAP Restricts Human Cytomegalovirus and Selectively Binds and Destabilizes Viral UL4/UL5 Transcripts

mBio ◽

10.1128/mbio.02683-20 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Ana Cristina Gonzalez-Perez ◽

Markus Stempel ◽

Emanuel Wyler ◽

Christian Urban ◽

Antonio Piras ◽

...

Keyword(s):

Gene Expression ◽

Zinc Finger ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Viral Infections ◽

Economic Losses ◽

Sequencing Analysis ◽

Antiviral Protein ◽

Hcmv Replication

ABSTRACT Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication. IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.

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Human Cytomegalovirus Drives WDR5 to the Virion Assembly Compartment to Facilitate Virion Assembly

10.1101/2020.08.03.235630 ◽

2020 ◽

Author(s):

Bo Yang ◽

YongXuan Yao ◽

Hui Wu ◽

Hong Yang ◽

Xue-Hui Ma ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Antiviral Treatment ◽

Critical Role ◽

Virion Assembly ◽

Potential Target ◽

Nuclear Egress ◽

Early Endosomes ◽

Hcmv Replication ◽

Assembly Compartment

AbstractWe previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WDR5 to facilitate capsid nuclear egress. Here, we further show that HCMV infection drives WDR5 to the perinuclear region by a mechanism that requires viral replication and intact microtubules. WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 interacted with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain the increasing WDR5 accumulation in the vAC during infection. WDR5 was then incorporated into HCMV virions and localized to the tegument layer, as demonstrated by fractionation and immune-gold electron microscopy. Thus, WDR5 is driven to the vAC and incorporated into virions, suggesting that WDR5 facilitates HCMV replication at later stage of virion assembly besides the capsid nuclear egress stage. These data highlight that WDR5 is a potential target for antiviral therapy.ImportanceHuman cytomegalovirus (HCMV) has a large (~235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are profoundly reconfigured to establish the virion assembly compartment (vAC), which is important for efficient assembly of progeny virions. We previously reported that WDR5 promotes HCMV nuclear egress. Here, we show that WDR5 is further driven to the vAC and incorporated into virions, perhaps to facilitate efficient virion maturation. This work identified potential roles for WDR5 in HCMV replication in the cytoplasmic stages of virion assembly. Taken together, WDR5 plays a critical role in HCMV capsid nuclear egress and is important for virion assembly, and thus is a potential target for antiviral treatment of HCMV-associated diseases.

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Detection of Human Cytomegalovirus in Different Histopathological Types of Glioma in Iraqi Patients

BioMed Research International ◽

10.1155/2015/642652 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Haidar A. Shamran ◽

Haider S. Kadhim ◽

Aws R. Hussain ◽

Abdulameer Kareem ◽

Dennis D. Taub ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Viral Infections ◽

Antigen Expression ◽

Active Role ◽

The Body ◽

Benign Meningioma ◽

Late Antigen ◽

Embedded Blocks

Human Cytomegalovirus (HCMV) is an endemic herpes virus that reemerges in cancer patients enhancing oncogenic potential. HCMV infection is associated with certain types of cancer morbidity such as glioblastomas. HCMV, like all other herpes viruses, has the ability to remain latent within the body of the host and can contribute in chronic inflammation. To determine the role of HCMV in glioma pathogenesis, paraffin-embedded blocks from glioma patients (n=50) and from benign meningioma patients (n=30) were obtained and evaluated by immunohistochemistry and polymerase chain reaction for the evidence of HCMV antigen expression and the presence of viral DNA. We detected HCMV antigen and DNA for IEI-72, pp65, and late antigen in 33/36, 28/36, and 26/36 in glioblastoma multiforme patients whereas 12/14, 10/14, and 9/14 in anaplastic astrocytoma patients, respectively. Furthermore, 84% of glioma patients were positive for immunoglobulin G (IgG) compared to 72.5% among control samples (P=0.04). These data indicate the presence of the HCMV virus in a high percentage of glioma samples demonstrating distinct histopathological grades and support previous reports showing the presence of HCMV infection in glioma tissue. These studies demonstrate that detection of low-levels of latent viral infections may play an active role in glioma development and pathogenesis.

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MicroRNA miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a

Journal of Virology ◽

10.1128/jvi.01740-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1070-1082 ◽

Cited By ~ 43

Author(s):

Ya-Ru Fu ◽

Xi-Juan Liu ◽

Xiao-Jun Li ◽

Zhang-zhou Shen ◽

Bo Yang ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Birth Defects ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Viral Gene Expression ◽

Viral Gene ◽

Gene Products ◽

Hcmv Replication

ABSTRACTCongenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products—IE1, pp71, and UL26—were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.IMPORTANCEHuman cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

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The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing

PLoS Pathogens ◽

10.1371/journal.ppat.1008807 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1008807

Author(s):

Einat Seidel ◽

Liat Dassa ◽

Corinna Schuler ◽

Esther Oiknine-Djian ◽

Dana G. Wolf ◽

...

Keyword(s):

Nk Cells ◽

Human Cytomegalovirus ◽

Cell Activation ◽

Hcmv Infection ◽

Nk Cell ◽

Innate Immune ◽

Target Cells ◽

Histocompatibility Complex ◽

Immunosuppressed Patients ◽

Infected Cells

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.

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Asparagine deprivation causes a reversible inhibition of Human Cytomegalovirus acute virus replication

10.1101/690016 ◽

2019 ◽

Author(s):

Chen-Hsuin Lee ◽

Samantha Griffiths ◽

Paul Digard ◽

Nhan T. Pham ◽

Manfred Auer ◽

...

Keyword(s):

Amino Acid ◽

Virus Replication ◽

Human Cytomegalovirus ◽

Late Onset ◽

Host Factors ◽

Prolonged Treatment ◽

Solid Organ ◽

Hcmv Disease ◽

Wide Range ◽

Hcmv Replication

AbstractAs obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High throughput siRNA screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes including the mediator complex, proteasome function and mRNA splicing. Focused characterisation of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus-1 or influenza A virus replication, suggesting the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued seven days post-infection with addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the non-essential amino acid, asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients.ImportanceHCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.

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Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1β Protein during Virion-Mediated Activation of the AIM2 Inflammasome

mBio ◽

10.1128/mbio.02510-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Sara Botto ◽

Jinu Abraham ◽

Nobuyo Mizuno ◽

Kara Pryke ◽

Bryan Gall ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Innate Immune ◽

Host Cells ◽

Interleukin 1Β ◽

Immediate Early ◽

Interferon Response ◽

Type I

ABSTRACTSecretion of interleukin-1β (IL-1β) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptorabsentinmelanoma 2 (AIM2) is required for secretion of IL-1β following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1β secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1β processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1β gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCEPersistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1β. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1β maturation to prevent this response, we find that HCMV and IE86 target the IL-1β protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1β transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.

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Nanotechnology against human cytomegalovirus in vitro: polyanionic carbosilane dendrimers as antiviral agents

Journal of Nanobiotechnology ◽

10.1186/s12951-021-00809-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

I. Relaño-Rodríguez ◽

M. S. Espinar-Buitrago ◽

V. Martín-Cañadilla ◽

R. Gómez-Ramirez ◽

J. L. Jiménez ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Viral Infections ◽

Cell Attachment ◽

Low Cost ◽

Antiviral Agents ◽

Carbosilane Dendrimers ◽

Potential Activity ◽

New Treatments

Abstract Background Human cytomegalovirus (HCMV) is a worldwide infection, causing different troublesome in immunosupressed patients and very related to Human Immunodeficiency Virus 1 (HIV-1) infection, mainly in developing countries, with a co-infection rate of 80% in Africa. The high cost of present treatments and the lack of routinely tests in these countries urge the necessity to develop new molecules or strategies against HCMV. The new treatments should be low-cost and capable of avoiding the emerging problem of resistant virus. Nanoparticles play an important role in several viral infections. Our main focus is to study the potential activity of polyanionic carbosilane dendrimers (PDC), which are hyperbranched molecules with several sulfonate or sulfate groups in their periphery, against different viruses. Results We studied the activity of G1-S4, G2-S16 and G2-S24P PDCs in MRC-5 cell line against HCMV infection by several plaque reduction assays. Our results show that dendrimers present good biocompatibility at the concentrations tested (1–50 µM) for 6 days in cell culture. Interestingly, both G2-S16 and G2-S24P showed a remarked inhibition at 10 µM against HCMV infection. Results on attachment and virucidal assays indicated that the inhibition was not directed to the virus or the virus-cell attachment. However, results of time of addition, showed a longer lasting activity of these dendrimers in comparison to ganciclovir, and the combination of G2-S16 or G2-S24P with ganciclovir increases the HCMV inhibition around 90 %. Conclusions Nanotechnology, in particular polyanionic carbosilane dendrimers, have proved their potential application against HCMV, being capable of inhibiting the infection by themselves or enhancing the activity of ganciclovir, the actual treatment. These compounds represent a low-cost approach to fight HCMV infections.

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Human cytomegalovirus infection among treatment-naive HIV-1 infected patients in Ethiopia

PLoS ONE ◽

10.1371/journal.pone.0247264 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0247264

Author(s):

Mulugeta Kiros ◽

Alene Geteneh ◽

Henok Andualem ◽

Derbie Alemu ◽

Abebech Tesfaye ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Addis Ababa ◽

Laboratory Diagnosis ◽

Conventional Pcr ◽

Hcmv Disease ◽

Treatment Naïve ◽

Hcmv Replication ◽

Human Cytomegalovirus Infection ◽

Hiv 1

Subclinical human cytomegalovirus (HCMV) replication is associated with immune dysfunction in immuno-suppressed antiretroviral therapy (ART) naive HIV infected individuals. No data is documented in Ethiopia so far concerning HCMV co-infection among HIV infected individuals. Hence, this study was aimed at generating data regarding the prevalence of active HCMV infection among treatment-naive HIV-infected individuals from Ethiopia. For this purpose, we enrolled 97 treatment-naive HIV infected study subjects in Addis Ababa from June to December 2018. ELISA and conventional PCR were performed consecutively to detect HCMV specific IgM antibody and HCMV DNA respectively. Of the 97 study subjects, 12 (12.4%) were positive for anti-CMV IgM antibodies but were not confirmed by PCR. With regard to the PCR positivity, 4/97 (4.1%) samples were positive for HCMV DNA. No statically significant associations were found between the dependent and independent variables. The presence of HCMV DNA in the current study highlights the need for a routine laboratory diagnosis for preventing HCMV disease among HIV-infected individuals early. Besides, the use of anti-CMV therapy for these CMV viremic individuals is also recommended as this can reduce the burden of CMV complications and consecutively prolonging the life of HIV infected individuals.

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Human Cytomegalovirus uses a Host Stress Response to Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very Long Chain Fatty Acids

10.1101/2021.01.16.426974 ◽

2021 ◽

Author(s):

Yuecheng Xi ◽

Lena Lindenmayer ◽

Ian Kline ◽

Jens von Einem ◽

John G. Purdy

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Lipid Metabolism ◽

Er Stress ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Lipid Synthesis ◽

Protein Levels ◽

Hcmv Replication ◽

Long Chain

ABSTRACTStress and virus infection are known to regulate lipid metabolism in cells. Human cytomegalovirus infection induces fatty acid (FA) elongation and increases the cellular abundance of lipids with very long-chain FA tails (VLCFAs). While reprogramming of metabolism can be stress-related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knockout PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. We found that in HCMV-infected cells, PERK promotes the increase in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFAs tails. Consistent with the SFA/MUFA lipidome changes, PERK enhances the protein levels of FA elongase 7 (ELOVL7), which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection was independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Consistent with the PUFA lipidome changes, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK—and more broadly, ER stress—may be necessary for membrane biogenesis needed to generate infectious HCMV virions.IMPORTANCEHCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator, PERK, controls fatty acid (FA) elongation and cellular abundance of several types of lipids following HCMV infection. Specifically, we found that PERK promotes FA elongase 7 synthesis of lipids with saturated/monounsaturated very long-chain FA tails which are important for building the viral membrane of infectious HCMV virions. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

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