Human Cytomegalovirus Replication Research Articles

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

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.

Intrinsic Immune Mechanisms Restricting Human Cytomegalovirus Replication.

Cellular restriction factors (RFs) act as important constitutive innate immune barriers against viruses. In 2006, the promyelocytic leukemia protein was described as the first RF against human cytomegalovirus (HCMV) infection which is antagonized by the viral immediate early protein IE1. Since then, at least 15 additional RFs against HCMV have been identified, including the chromatin regulatory protein SPOC1, the cytidine deaminase APOBEC3A and the dNTP triphosphohydrolase SAMHD1. These RFs affect distinct steps of the viral replication cycle such as viral entry, gene expression, the synthesis of progeny DNA or egress. This review summarizes our current knowledge on intrinsic immune mechanisms restricting HCMV replication as well as on the viral strategies to counteract the inhibitory effects of RFs. Detailed knowledge on the interplay between host RFs and antagonizing viral factors will be fundamental to develop new approaches to combat HCMV infection.

HCMV infection and IFITM3 rs12252 are associated with Rasmussen's encephalitis disease progression.

ObjectiveRasmussen’s encephalitis (RE) is a rare and severe progressive epileptic syndrome with unknown etiology. Infection by viruses such as human cytomegalovirus (HCMV) has been hypothesized to be a potential trigger for RE. Interferon‐induced transmembrane protein‐3 (IFITM3) single‐nucleotide polymorphism (SNP) rs12252 is associated with the severity of viral infection disease. This study aimed to address the possibility that HCMV infection and IFITM3 rs12252 might be associated with RE disease progression.MethodsThe expression of HCMV and IFITM3 was detected with immunohistochemical staining, in situ hybridization and immunofluorescence double staining. The genotype of IFITM3 rs12252 was detected using the Sanger sequencing method. A genetic association analysis was carried out for this SNP and HCMV antigen expression. The relationship between this SNP and the clinical characteristics of these patients was further analyzed. In in vitro study, HCMV replication in SH‐SY5Y cells with overexpressed IFITM3 variant was detected by immunofluorescence and real‐time RT‐PCR.ResultsElevated expression of HCMV and IFITM3 was observed in the brain tissue of RE patients. Moreover, the IFITM3 polymorphism rs12252‐C was found to associate with HCMV high detection and rapid disease progression in RE patients with the IFITM3 rs12252‐CC genotype. In vitro study showed the overexpressed IFITM3 variant was associated with HCMV high infection level.ConclusionThese results suggest that the IFITM3 rs12252‐C is associated with the disease progression of RE patients via facilitating persistent HCMV infection in brain tissue and provides new insight into understanding the pathogenesis of RE.

Stable and Inducible Gene Knockdown in Primary Human Fibroblasts: A Versatile Tool to Study the Role of Human Cytomegalovirus Host Cell Factors.

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.

Using CRISPR technology to inhibit the replication of human cytomegalovirus by deletion of a gene promoter

Human cytomegalovirus (HCMV) causes serious infections in immunocompromised patients. It is ubiquitous in nature and has the potential to be transmitted via non-sexual contact (saliva, breast-feeding, placental transfer, blood transfusion, organ transplantation) or sexual contact. Although HCMV infection resolves spontaneously in immunocompetent hosts, it causes serious life-threatening disease in immunocompromised patients and can reactivate from latency at any time during life when the host’s immune system is compromised. Management of these infections is complex as currently available antiviral agents have several limitations, and thus mortality of these infections approaches 50%. Reactivation of latent HCMV remains a major concern and is associated with a high morbidity and mortality in high-risk immunocompromised adults. Although great progress has been made in the management of HCMV disease over the last several decades, management options are still limited due to major issues with dosing and side effects. Also, none of these medications inhibit latent HCMV lying dormant in cells. Gene-based targeting mechanisms have the added advantages of inhibiting not only the reactivated HCMV but also the latent HCMV in cells, as well as stopping the dysregulation of innate immunity occurring during the early stages of HCMV infection. Using CRISPR/Cas9 technology/synthetic single gRNA, site-specific cleavage was carried out with successful deletion of a major intermediate early gene promoter/enhancer (MIEP/E) of HCMV. This study demonstrated the efficacy of the sgRNA-CRISPR/Cas9 strategy to delete the MIEP/E of HCMV. Deletion of this promoter is expected to block the expression of early and late phase genes, inhibit HCMV replication, prevent latency, and protect the cellular immune system, in immunocompromised patients.

Type I IFNs: A Blessing in Disguise or Partner in Crime in MERS-CoV-, SARS-CoV-, and SARS-CoV-2-Induced Pathology and Potential Use of Type I IFNs in Synergism with IFN-γ as a Novel Antiviral Approach Against COVID-19.

Since the end of 2019, the emergence of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accelerated the research on host immune responses toward the coronaviruses. When there is no approved drug or vaccine to use against these culprits, host immunity is the major strategy to fight such infections. Type I interferons are an integral part of the host innate immune system and define one of the first lines of innate immune defense against viral infections. The in vitro antiviral role of type I IFNs against Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV (severe acute respiratory syndrome coronavirus) is well established. Moreover, the involvement of type I IFNs in disease pathology has also been reported. In this study, we have reviewed the protective and the immunopathogenic role of type I IFNs in the pathogenesis of MERS-CoV, SARS-CoV, and SARS-CoV-2. This review will also enlighten the potential implications of type I IFNs for the treatment of COVID-19 when used in combination with IFN-γ.

The role of IFN-gamma in Humoral immune response for HCMV antigens among pregnant women

Background: Human cytomegalovirus (HCMV) is a highly host-specific virus belongs to the βherpesvirus subfamily, which is a leading cause of congenital infections. The immune cytokines such as Interferons-gama (IFN- ) may have direct inhibitory effects on HCMV replication and control of viral infections. Aim of the study: This research aims at determining the specificity of anti-HCMV antibodies for different HCMV antigens in relation to serum INF- levels. Materials & Methods: A cross sectional study was carried out in Kirkuk governorate from April 2018 to June 2019. The number of pregnant women understudy was 400 women presented to some private medical laboratories. The pregnant women were examined for the seroprevalence of HCMVIgM and IgG by using ECLIA technique then their specificities determined for different HCMV antigens by using line immune assay, In addition to estimation the level of serum INF- levels by using ELISA technique. Results: The rate of HCMV-IgG , HCMV-IgM and both HCMV-IgG and IgM at the same time were 288(72 %), 32(8%) and 18(4.5%) respectively. Regarding the specificity of the determined HCMVIgM to various HCMV antigens (IE1, CM2, p150, p65, gB1 and gB2), the highest rate of HCMVIgG was 96.25% specific for gB1 antigen, while highest rate of HCMV-IgM were 96.87% specific gB1 and p150 antigen. Considering the specificity of these antibodies for the examined antigens in relation to serum INF- levels, the highest of pregnant women with increased INF- level had antibodies for IE1 antigen. Conclusions: There was significant relation of the serum INF- level with specificity of anti-HCMV antibodies to divers HCMV antigens.

The role of autophagy in human cytomegalovirus IE2 expression.

The purpose of this study was to determine whether autophagy regulates the expression of human cytomegalovirus (HCMV) immediately early two viral protein (IE2). Rapamycin and 3-methyladenine (3-MA) were used to stimulate or suppress autophagy during HCMV infection. UL122 recombinant plasmid was transfected to overexpress IE2 and small interference RNA against autophagy-related protein 3 (ATG3) was used to knockdown ATG3. Western blot was performed to measure the expression of viral proteins and autophagy levels. Immunofluorescence was used to detect the immediately early 1 viral protein (IE1) expression. In human embryonic lung fibroblasts, infection of HCMV promotes the lipidation of light chain 3 (LC3) at 6 and 24 hours post infection (hpi), which was accompanied by the increased expression of viral protein IE2. When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection, the autophagy hallmarks LC3II and ATG3 were upregulated. Furthermore, viral protein IE2 expression was reduced at 24 and 48 hpi either by the treatment of autophagy inducer rapamycin or by the inhibitor 3-MA before HCMV infection. At the same time, small interference ATG3 transient transfection, used to suppress autophagy, significantly inhibited IE2 expression. However, when 3-MA was used to regulate autophagy levels after HCMV infection, expression of IE2 and IE1 were both decreased, while autophagy inducer rapamycin treatment after HCMV infection increased IE2 expression slightly. IE2 was involved in autophagy induced by HCMV infection and blocking autophagy could inhibit the expression of HCMV viral protein IE2, which might be one way for autophagy to restrict HCMV replication.

Synthesis of New Imidazopyridine Nucleoside Derivatives Designed as Maribavir Analogues.

The strong inhibition of Human Cytomegalovirus (HCMV) replication by benzimidazole nucleosides, like Triciribine and Maribavir, has prompted us to expand the structure–activity relationships of the benzimidazole series, using as a central core the imidazo[4,5-b]pyridine scaffold. We have thus synthesized a number of novel amino substituted imidazopyridine nucleoside derivatives, which can be considered as 4-(or 7)-aza-d-isosters of Maribavir and have evaluated their potential antiviral activity. The target compounds were synthesized upon glycosylation of suitably substituted 2-aminoimidazopyridines, which were prepared in six steps starting from 2-amino-6-chloropyridine. Even if the new compounds possessed only a slight structural modification when compared to the original drug, they were not endowed with interesting antiviral activity. Even so, three derivatives showed promising cytotoxic potential.

Artemisinins target the intermediate filament protein vimentin for human cytomegalovirus inhibition

The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentin-deficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virus-induced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti-HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin-binding sites and compound moieties for binding.

The Clinically Approved Antifungal Drug Posaconazole Inhibits Human Cytomegalovirus Replication.

Posaconazole (PCZ) is a clinically approved drug used predominantly for prophylaxis and salvage therapy of fungal infections. Here, we report its previously undescribed anti-human cytomegalovirus (HCMV) activity. By using antiviral assays, we demonstrated that PCZ, along with other azolic antifungals, has a broad anti-HCMV activity, being active against different strains, including low-passage-number clinical isolates and strains resistant to viral DNA polymerase inhibitors. Using a pharmacological approach, we identified the inhibition of human cytochrome P450 51 (hCYP51), or lanosterol 14α demethylase, a cellular target of posaconazole in infected cells, as a mechanism of anti-HCMV activity of the drug. Indeed, hCYP51 expression was stimulated upon HCMV infection, and the inhibition of its enzymatic activity by either the lanosterol analog VFV {(R)-N-(1-(3,4'-difluoro-[1,1'-biphenyl]-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide} or PCZ decreased HCMV yield and infectivity of released virus particles. Importantly, we observed that the activity of the first-line anti-HCMV drug ganciclovir was boosted tenfold by PCZ and that ganciclovir (GCV) and PCZ act synergistically in inhibiting HCMV replication. Taken together, these findings suggest that this clinically approved drug deserves further investigation in the development of host-directed antiviral strategies as a candidate anti-HCMV drug with a dual antimicrobial effect.

The host exosome pathway underpins biogenesis of the human cytomegalovirus virion.

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constituents. To validate this acquisition, we characterized exosomes released from uninfected cells, and demonstrated that over 99% of the protein cargo was subsequently incorporated into HCMV virions during infection. This suggested a common membrane origin, and utilization of host exosome machinery for virion assembly and egress. Thus, we selected a panel of exosome proteins for knock down, and confirmed that loss of 7/9 caused significantly less HCMV production. Saliently, we report that VAMP3 is essential for viral trafficking and release of infectious progeny, in various HCMV strains and cell types. Therefore, we establish that the host exosome pathway is intrinsic for HCMV maturation, and reveal new host regulators involved in viral trafficking, virion envelopment, and release. Our findings underpin future investigation of host exosome proteins as important modulators of HCMV replication with antiviral potential.

Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early 1 gene.

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

Articles of Significant Interest in This Issue

Host cells exhibit a wide array of responses upon infection to limit pathogen replication efficiency. Nukui et al. (e00033-20) found that posttranslationally modifying key viral proteins via S-nitrosylation limits human cytomegalovirus (HCMV) replication efficiency. The viral protein pp71 inhibits the potent STING antiviral pathway, thereby reducing subsequent innate responses. However, protein S-nitrosylation of pp71 limits the capacity of this viral protein to undermine the host STING pathway, providing an additional level of antiviral response to infection.

The Artemisinin-Derived Autofluorescent Compound BG95 Exerts Strong Anticytomegaloviral Activity Based on a Mitochondrial Targeting Mechanism.

Human cytomegalovirus (HCMV) is a major human pathogen associated with severe pathology. Current options of antiviral therapy only partly satisfy the needs of a well-tolerated long-term treatment/prophylaxis free from drug-induced viral resistance. Recently, we reported the strong antiviral properties in vitro and in vivo of the broad-spectrum anti-infective drug artesunate and its optimized derivatives. NF-κB signaling was described as a targeting mechanism and additional target proteins have recently been identified. Here, we analyzed the autofluorescent hybrid compound BG95, which could be utilized for intracellular visualization by confocal imaging and a tracking analysis in virus-infected primary human fibroblasts. As an important finding, BG95 accumulated in mitochondria visualized by anti-prohibitin and MitoTracker staining, and induced statistically significant changes of mitochondrial morphology, distinct from those induced by HCMV infection. Notably, mitochondrial membrane potential was found substantially reduced by BG95, an effect apparently counteracting efficient HCMV replication, which requires active mitochondria and upregulated energy levels. This finding was consistent with binding properties of artesunate-like compounds to mitochondrial proteins and thereby suggested a new mechanistic aspect. Combined, the present study underlines an important role of mitochondria in the multifaceted, host-directed antiviral mechanism of this drug class, postulating a new mitochondria-specific mode of protein targeting.

Peroxynitrite is essential for the initiation of cytomegalovirus replication in vitro and in vivo

Human cytomegalovirus (HCMV) is a pathogenic beta-herpesvirus that establishes a lifelong infection in hosts. It causes significant morbidity and mortality in the immunocompromised and is associated with a range of birth defects following congenital infection. Current therapeutic approaches that target key viral proteins are toxic and antiviral drug resistance is common. Thus, targeting host genes and cellular pathways essential for HCMV infection offers an alternative strategy for the development of antivirals. Here we show that host oxidative/nitrosative stress responses to CMV are critical for virus replication. Oxidative/nitrosative stress occurs due to accumulation of reactive oxygen/nitrogen species (ROS/RNS). Using a range of ROS/RNS scavengers, we identified that peroxynitrite, a powerful oxidant and nitrating agent, dramatically promoted virus replication in both in vitro and in vivo models of CMV infection. HCMV rapidly induced production of intracellular peroxynitrite upon infection. Inhibition of peroxynitrite within the first 24 hours alleviates efficient HCMV infection in both cell-free and cell-associated infection systems, indicating that peroxynitrite may influence pathways necessary for HCMV entry and/or replication. Furthermore, peroxynitrite inhibition also inhibited HCMV reactivation from latency. Interestingly, the neurotransmitter and naturally-occurring peroxynitrite antagonist 5-hydroxytryptamine, commonly known as serotonin, also impinged on HCMV-induced peroxynitrite production and exhibited anti-HCMV activity. Thus, overall, our study demonstrates a novel role for intracellular peroxynitrite in CMV pathogenesis and implies that peroxynitrite could be targeted as a novel approach to inhibiting CMV infection.

Target verification of artesunate-related antiviral drugs: Assessing the role of mitochondrial and regulatory proteins by click chemistry and fluorescence labeling

Human cytomegalovirus (HCMV) infection is associated with serious pathology such as transplant rejection or embryonic developmental defects. Antiviral treatment with currently available drugs targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. For this reason, novel ways of anti-HCMV therapy focusing on so far unexploited small molecules, targets and mechanisms are intensively studied. Recently, we described the pronounced antiviral activity of the artesunate-related class of trioxane compounds, comprising NF-κB/signaling inhibitors like the trimeric derivative TF27, which proved to be highly active in a nanomolar range both in vitro and in vivo. Here, we extend this analysis by presenting further TF27/artesunate-derived antiviral compounds designed for their specific use in target verification by click chemistry applied in fluorescence labeling and tag affinity strategies. Our main findings are as follows: (i) compounds TF27, BG95, AC98 and AC173 exert strong inhibitory activity against HCMV replication in cultured primary human cells, (ii) autofluorescence activity could be quantitatively detected for BG95 and AC98, and confocal fluorescence imaging revealed accumulation in mitochondria, (iii) postulated cellular targets including mitochondrial proteins were down-regulated upon TF27 treatment, (iv) a click chemistry-based protocol of target enrichment was established, and (v) mass spectrometry-based proteomic analysis, using proteins from HCMV-infected fibroblasts covalently interacting with AC173, revealed a refined list of targets. Combined, data strongly suggest a complex mode of antiviral drug-target interaction of artesunate-related compounds, now highlighting potential roles of mitochondrial, NF-κB pathway proteins, exportins and possibly more. This strategy may further promote antiviral drug development on the basis of pharmacologically optimized trioxane derivatives.

Targeting human cytomegalovirus IE genes by CRISPR/Cas9 nuclease effectively inhibits viral replication and reactivation.

Human cytomegalovirus (HCMV) infection causes high morbidity and mortality among immunocompromised patients and can remain in a latent state in host cells. Expression of the immediate-early (IE) genes sustains HCMV replication and reactivation. As a novel genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been extensively utilized to modify and edit genomic DNA. In the present study, the CRISPR/Cas9 system was used to target the IE region of the HCMV genome via specific single-guide RNAs (sgRNAs). Infection with CRISPR/Cas9/sgRNA lentiviral constructs significantly reduced viral gene expression and virion production in HFF primary fibroblasts and inhibited viral DNA production and reactivation in the THP-1 monocytic cell line. Thus, the CRISPR/Cas9/sgRNA system can accurately and efficiently target HCMV replication and reactivation and represents a novel therapeutic strategy against latent HCMV infection.

Antiviral Activities of Quercetin and Isoquercitrin Against Human Herpesviruses.

We previously reported that the ethyl acetate (EtOAc) fraction of a 70% ethanol extract of Elaeocarpus sylvestris (ESE) inhibits varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) replication in vitro. PGG (1,2,3,4,6-penta-O-galloyl-ß-D-glucose) is a major chemical constituent of the EtOAc fraction of ESE that inhibits VZV but not HCMV replication. In this study, we comprehensively screened the chemical compounds identified in the EtOAc fraction of ESE for potential antiviral properties. Among the examined compounds, quercetin and isoquercitrin displayed potent antiviral activities against both VZV and HCMV with no significant cytotoxic effects. Both compounds strongly suppressed the expression of VZV and HCMV immediate–early (IE) genes. Our collective results indicated that, in addition to PGG, quercetin and isoquercitrin are bioactive compounds in the EtOAc fraction of ESE that effectively inhibit human herpesvirus replication.

The peptidyl-prolyl cis/trans isomerase Pin1 interacts with three early regulatory proteins of human cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen of high clinical relevance. Despite intensive research of virus-host interaction, crucial details still remain unknown. In this study, the role of the cellular peptidyl-prolyl cis/trans isomerase Pin1 during HCMV infection was investigated. Pin1 is able to recognize phosphorylated serine/threonine-proline motifs and regulates the structural conformation, stability and function of its substrates. Concerning HCMV replication, our recent studies revealed that Pin1 plays an important role in viral nuclear egress by contributing to the depletion of the nuclear lamina at distinct sites through the cis/trans conversion of lamin proteins. Here, novel data illustrate the HCMV-induced upregulation of Pin1 including various cell types being permissive, semi-permissive or non-permissive for productive HCMV replication. Addressing the question of functional impact, Pin1 knock-out (KO) did not show a measurable effect on viral protein expression, at least when assessed by Western blot analysis. Applying highly sensitive methods of qPCR and plaque titration, a pharmacological inhibition of Pin1 activity, however, led to a significant decrease of viral genome equivalents and production of infectious virus, respectively. When focusing on the identification of viral proteins interacting with Pin1 by various coimmunoprecipitation (CoIP) settings, we obtained positive signals for (i) the core nuclear egress complex protein pUL50, (ii) the viral mRNA export factor pUL69 and (iii) the viral DNA polymerase processivity factor pUL44. Confocal immunofluorescence analysis focusing on partial colocalization between Pin1 and the coexpressed viral proteins pUL50, pUL69 or pUL44, respectively, was consistent with the CoIP experiments. Mapping experiments, using transient expression constructs for a series of truncated protein versions and specific replacement mutants, revealed a complex pattern of Pin1 interaction with these three early regulatory HCMV proteins. Data suggest a combination of different modes of Pin1 interactions, involving both classical phosphorylation-dependent Pin1 binding motifs and additional phosphorylation-independent binding sites. Combined, these results support the concept that Pin1 may play an important role in several stages of HCMV infection, thus determining viral replicative efficiency.