Divide et impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited HCMV AD169 strain in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.     

Late emergence of A594V and L595W mutations related to ganciclovir resistance in a patient with HCMV retinitis and long-term HIV progression

The emergence of ganciclovir (GCV) resistance during the treatment of human cytomegalovirus (HCMV) infection is a serious clinical challenge, and is associated with high morbidity and mortality. In this case report, we describe the emergence of two consecutive mutations (A594V and L595W) related to GCV resistance in a patient with HCMV retinitis and long-term HIV progression after approximately 240 days of GCV use. Following the diagnosis of retinitis, the introduction of GCV did not result in viral load reduction. The detected mutations appeared late in the treatment, and we propose that other factors (high initial HCMV load, previous GCV exposure, low CD4⁺ cell count), in addition to the presence of resistance mutations, may have contributed to the treatment failure of HCMV infection in this patient.     

巨细胞病毒DNA动态监测在预防肾移植术后巨细胞病毒性肺炎中的应用价值

目的 探讨荧光定量PCR动态监测在预防肾移植术后人巨细胞病毒(HCMV)肺炎中的临床应用价值.方法 同种异体肾移植患者242例.男144例,女98例,平均年龄42(17～71)岁.随机分为2组:实验组127例,对照组115例.实验组患者移植术后采用荧光定量PCR方法动态检测患者血、尿标本HCMV-DNA,其中任何一项HCMV-DNA拷贝数>1×103拷贝/ml者,连续静脉滴注更昔洛韦4周,按照肌酐清除率计算剂量(肾功能正常者剂量为5 mg/kg 2次/d;肾功能减退者,肌酐清除率50～69 ml/min时2.5 mg/kg,2次/d;肌酐清除率25～49 ml/min时2.5 mg/kg,2次/d;肌酐清除率10～24 ml/min时1.25 mg/kg,1次/d;肌酐清除率<10 ml/min时每周3次每次1.25 mg/kg,于血液透析后给予).对照组不进行定期检测及更昔洛韦预防用药,比较2组患者HC-MV肺炎发病、治疗情况及移植肾1年存活率. 结果实验组术后HCMV肺炎发生率6.3%(8/127);肺炎发生中位时间84(46～167)d;住院治疗中位时间36(30～57)d;病死率12.5%(1/8);呼吸机使用率12.5%(1/8),合并其他病原体感染率25.0%(2/8);移植肾1年存活率98.4%(125/127),其中1例为移植肾带功能死亡,1例为移植肾急性排斥失功.对照组术后HCMV肺炎发生率14.8%(17/115);肺炎发生中位时间51(34～138)d;住院中位时间40(21～67)d;病死率23.5%(4/17);呼吸机使用率29.4%(5/17),合并其他病原体感染率41.2%(7/17);移植肾1年存活率93.0%(107/115),死亡4例中3例为移植肾带功能,1例移植肾功能未恢复;4例为移植肾急性排斥失功.2组间比较住院治疗时间差异无统计学意义(P>0.05),其余各项(HCMV肺炎发生率、发生时间、病死率、呼吸机使用率、合并其他病原体感染率、移植肾1年存活率)差异均有统计学意义(P<0.05).结论 荧光定量PCR动态监测肾移植术后患者血、尿标本HCMV-DNA载量,预防术后HCMV肺炎效果好,移植肾1年存活率提高.     

Comparative sequence analysis of US28 gene of human cytomegalovirus strains isolated from HIV-positive patients

Human Cytomegalovirus (HCVM) encodes several G-protein coupled receptors, such as US28 protein. We determined the US28 gene sequence from clinical isolates obtained from 17 Human Immunodeficiency Virus (HIV)-infected patients with HCMV infection. Two types of clinical specimens were collected : peripheral blood leucocytes (PBLs) from 12 patients with HCMV-positive viremia and cerebro-spinal fluids (CSF) from five patients with HCMV-encephalitis. Comparison of US28 nucleotide sequences between clinical specimens and several HCMV reference strains showed that mutations were clustered at both ends of the gene. The mutations observed at the C-terminus were observed at some sequences whereas the mutations observed at the N-terminus lead us to define five patterns of mutations. These patterns were equally distributed among isolates obtained from CSF or PBLs of HIV-infected patients. For each clinical isolate, the gB genotype was determined. There was no genetic association between gB genotype and US28 patterns. The comparison of the results of the present study and those published from sequences obtained from children with HCMV congenital disease (Arav-Boger, 2002) demonstrates that two motifs appear especifically either in US28 protein sequences obtained from HIV-positive patients (motif 4) or in US28 protein sequences obtained from patients with congenital HCMV infection (motif 5/genotype B). The appearance of these patterns of mutations in other clinical context needs to be studied.     

STING facilitates nuclear import of herpesvirus genome during infection

Once inside the host cell, DNA viruses must overcome the physical barrier posed by the nuclear envelope to establish a successful infection. The mechanism underlying this process remains unclear. Here, we show that the herpesvirus exploits the immune adaptor stimulator of interferon genes (STING) to facilitate nuclear import of the viral genome. Following the entry of the viral capsid into the cell, STING binds the viral capsid, mediates capsid docking to the nuclear pore complex via physical interaction, and subsequently enables accumulation of the viral genome in the nucleus. Silencing STING in human cytomegalovirus (HCMV)-susceptible cells inhibited nuclear import of the viral genome and reduced the ensuing viral gene expression. Overexpressing STING increased the host cell’s susceptibility to HCMV and herpes simplex virus 1 by improving the nuclear delivery of viral DNA at the early stage of infection. These observations suggest that the proviral activity of STING is conserved and exploited by the herpesvirus family. Intriguingly, in monocytes, which act as latent reservoirs of HCMV, STING deficiency negatively regulated the establishment of HCMV latency and reactivation. Our findings identify STING as a proviral host factor regulating latency and reactivation of herpesviruses.

Human cytomegalovirus (HCMV) belongs to the β-herpesvirus subfamily and establishes lifelong latent infection in 60 to 90% of the population worldwide. The inability of the immune response to clear the virus and the absence of a vaccine renders HCMV a serious global health burden (1). HCMV is the most common cause of congenital viral infection during pregnancy and also increases the morbidity and mortality among immunosuppressed transplant patients and AIDS patients (2, 3).DNA viruses must sequentially overcome two physical barriers, the plasma membrane and the nuclear envelope, to replicate in the nucleus (4). The herpesvirus double-stranded DNA (dsDNA) genome is tightly packed inside an icosahedral capsid structure measuring 130 nm in diameter (5, 6). The nuclear pore complex (NPC) allows the transport of macromolecules up to 39 nm in diameter; thus, nuclear entry of herpesviruses with a capsid diameter that is larger than this diffusion limit is a critical step for successful viral infection (7). Herpesviruses employ an uncoating strategy whereby the capsid undergoes conformational change without disruption of the capsid structure, open the portal, and release the viral DNA into the nucleus via NPC. Several host cell surface receptors determining HCMV cell tropism and permissiveness have been identified (8–14); however, the host factor(s) involved in capsid uncoating and nuclear genome import is (are) yet to be uncovered.In the event of improper translocation of the viral genome, the genome is exposed to the cytoplasm. Then, host factors recognize viral DNA as a foreign antigen and elicit antiviral immune response resulting in degradation of the viral genome by cytosolic nucleases (15). Pressure-driven viral DNA release into the host nucleus accounts for only 50% of DNA ejection into the cellular environment, and small polycationic molecules can block viral genome ejection from the capsid, according to recent studies (16, 17). These findings offer two interesting perspectives: the presence of unknown host factors that regulate uncoating and the possibility of developing antiviral agents that target this uncoating process.Although a primary lytic HCMV infection induces robust innate and adaptive immune responses, HCMV is not completely cleared in vivo but, rather, persists in the host cell, becoming a lifelong potential threat (18). HCMV has contrived a strategy to establish latency in the host cell, such as CD34⁺ hematopoietic progenitor cell (HPC) and CD14⁺ monocyte, to survive. Although several entry receptors in fibroblasts and endothelial and epithelial cells have been identified (8–14), only a few host genes or signaling pathways in monocytic cells required for HCMV infection have been researched. For example, EGFR and c-Src signaling must be active to lead to cytoplasmic trafficking, nuclear translocation, and infection in monocytes (19, 20). However, host factors that regulate the HCMV latency and reactivation after HCMV enters the monocytes are largely unknown.Stimulator of interferon (IFN) genes (STING) is an endoplasmic reticulum–resident membrane protein ubiquitously expressed in both immune cells and nonimmune cells (21, 22). Recognition of cytosolic dsDNA leads to the synthesis of cyclic GMP–AMPs (cGAMPs) by activating a cyclic GMP–AMP synthase (cGAS). cGAMPs bind to STING, inducing activation of TBK1 and IRF3 signaling cascades (23). The canonical IFN-dependent role of STING in antiviral immunity has been extensively investigated. Interestingly, several recent reports suggest a noncanonical proviral function of STING in viral infection (24, 25). A potential proviral role of STING in HCMV infection is largely unknown.Here, we demonstrate a proviral function of STING during herpesvirus infection. We show that ectopic expression of STING in STING-deficient cells converts an otherwise insusceptible cell to an HCMV- and herpes simplex virus 1 (HSV-1)–susceptible cell, implying that this STING function is evolutionarily conserved. Furthermore, loss of STING significantly represses the establishment of HCMV latency and viral reactivation in monocytic cells, suggesting that STING is a host factor that regulates the herpesvirus life cycle.     

Viral escape from NK-cell-mediated immunosurveillance: A lesson for cancer immunotherapy?

Natural killer (NK) cells are innate lymphocytes that participate in immune responses against virus-infected cells and tumors. As a countermeasure, viruses and tumors employ strategies to evade NK-cell-mediated immunosurveillance. In this review, we examine immune evasion strategies employed by viruses, focusing on examples from human CMV and severe acute respiratory syndrome coronavirus 2. We explore selected viral evasion mechanisms categorized into three classes: (1) providing ligands for the inhibitory receptor NKG2A, (2) downregulating ligands for the activating receptor NKG2D, and (3) inducing the immunosuppressive cytokine transforming growth factor (TGF)-β. For each class, we draw parallels between immune evasion by viruses and tumors, reviewing potential opportunities for overcoming evasion in cancer therapy. We suggest that in-depth investigations of host–pathogen interactions between viruses and NK cells will not only deepen our understanding of viral immune evasion but also shed light on how NK cells counter such evasion attempts. Thus, due to the parallels of immune evasion by viruses and tumors, we propose that insights gained from antiviral NK-cell responses may serve as valuable lessons that can be leveraged for designing future cancer immunotherapies.     

CD45RA expression on HCMV-specific effector memory CD8+ T cells is associated with the duration and intensity of HCMV replication after transplantation

In this cross-sectional study on 42 solid organ transplant recipients, the association of kinetics of human cytomegalovirus (HCMV) replication and EMRA HCMV-specific CD8+ T cells was investigated. Correlation was observed between the duration of HCMV replication after transplantation and CD45RA+CD27− (r = 0.609; p = 0.004), CD45RA+ CD28− (r = 0.579; p = 0.008) or CD45RA+CCR7− (r = 0.488; p = 0.029) HCMV-specific CD8+ T cells percentages. In the multivariate regression analyses, CD45RA+CD27−, CD45RA+CD28− or CD45RA+CCR7− HCMV-specific CD8+ T cells percentages increased 5.58% (p = 0.001), 5.35% (p = 0.001) or 4.49% (p = 0.012), respectively, with every 10-day increase in the duration of HCMV replication. Moreover, CD45RA+CD27− or CD45RA+CD28− frequencies increased 4.16% (p = 0.024) or 3.58% (p = 0.049), respectively, with every unity increase in log₁₀ genomes/mL. These observations support the major association between the frequency of EMRA HCMV-specific CD8+ T cells and the duration of post-transplant HCMV replication episodes in solid organ transplantation recipients.     

Ex vivo monitoring of human cytomegalovirus‐specific CD8+ T‐Cell responses using the QuantiFERON®‐CMV assay in allogeneic hematopoietic stem cell transplant recipients attending an Irish hospital

Reconstitution of human cytomegalovirus (HCMV) T‐cell immunity is crucial in hematopoietic stem cell transplant (HSCT) recipients. The QuantiFERON®‐CMV assay for cellular HCMV‐specific immunity was evaluated in allogeneic HSCT recipients (n = 43) and patients with hematological malignancies (n = 29) attending a tertiary‐care Irish hospital. An intracellular cytokine (ICC) assay correlated with the QuantiFERON®‐CMV assay. Although there was agreement between HCMV seropositivity and QuantiFERON®‐CMV assay, six HCMV seropositive immunosuppressed patients with hematological malignancy had negative QuantiFERON®‐CMV results. The 43 HSCT recipients were classified as high risk (D^?/R⁺) (n = 18), intermediate risk (D⁺/R⁺ and D⁺/R^?) (n = 17), and low risk (D^?/R^?) (n = 8). During episodes of HCMV DNAemia no evidence of HCMV‐specific immunity was found using the QuantiFERON®‐CMV assay. Furthermore, the recovery of HCMV‐specific CD8⁺ T‐cell responses in high‐risk seropositive recipients of matched unrelated donors was severely delayed, a mean of 200 (SD = 117) days compared to 58 (SD = 23) days for sibling donors (P ≤ 0.028). In addition, three patients with late HCMV infection (infection >100 days post‐transplant) had delayed reconstitution of HCMV‐specific CD8⁺ T cells. Interestingly, two recipients (R⁺/D^?) developed rapid immune reconstitution by days 15 and 36 post‐HSCT, suggesting HCMV‐specific T‐cell lymphopoiesis of recipient origin. Levels of CD8⁺ T‐cell immunity in HCMV seropositive HSCT recipients were lowest following HSCT. A high number (33%) of indeterminate results was observed immediately after transplantation. Patients with indeterminate QuantiFERON®‐CMV results had low levels of HCMV‐specific CD8⁺ T cells. J. Med. Virol. 82:433–440, 2010. © 2010 Wiley‐Liss, Inc.     

人巨细胞病毒PP65_(322～561)片段的原核表达及功能鉴定

目的 构建人巨细胞病毒(HCMV)PP65_(322～561)蛋白片段的原核表达载体PQE30-PP65,并表达蛋白.方法 PCR扩增HCMV PP65第976～1686位核苷酸片段;构建重组表达质粒PQE30-PP65转化M15并表达蛋白;PCR、SDS-PAGE及Weatern blot鉴定重组载体及表达产物;ELISA检测目的蛋白的抗原性.结果构建了PQE30-PP65表达载体,在M15菌株中成功表达PP65_(322～561)蛋白,蛋白浓度达到2.2g/t.并与pp65单克隆抗体及HCMV LgG阳性血清反应良好.结论 成功构建PQE30-PP65表达载体,表达的目的蛋白具有良好的抗原性.     

应用原位免疫聚合酶链反应检测活性巨细胞病毒感染

目的 建立一种新的诊断活动性巨细胞病毒感染的方法。方法 应用原位免疫聚合酶链反应（PCR）技术检测早、中、晚期孕妇宫颈脱落细胞中巨细胞病毒抗原。结果 119例受检标本中16例出现人巨细胞病毒（HCMV）抗原阳性，孕早、中、晚期孕妇活动性HCMV感染率分别为：9．37％、11．90％、17．77％；检出的HCMV抗原阳性细胞数为2－38／5万宫颈脱落细胞，平均8．5／5万细胞。常规ABC法平行检测，原位免疫PCR法检测敏感性高于ABC法。结论 原位免疫PCR方法敏感性高、特异性强，可检出测量HCMV抗原，为临床诊断活动性HCMV感染提供一种新方法。