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.     

HCMV pp65 DNA疫苗的纯化与检定

目的研究HCMV pp65 DNA疫苗（pcDNA3.1-pp65）的纯化工艺,并建立质控标准。方法对工程菌（DH5α/pcDNA3.1-pp65）的高效发酵菌体采用碱裂解法进行质粒初提;三步柱层析（依次为分子筛层析、亲和层析、阴离子层析）对质粒进行纯化,检测质粒含量、超螺旋比例、内毒素等,最后对终产品质粒溶液进行全面质量检定。结果质粒初提液通过分子筛层析后,去除了大量RNA、宿主DNA、内毒素等,再经亲和柱纯化后获得了高比例的超螺旋质粒DNA,达95%,经阴离子柱层析有效去除内毒素,质粒得率为0.8-0.95 mg/g菌,质粒总回收率达72.7%。三批终产品全面质量检定均符合规定。结论建立了稳定的HCMV pp65 DNA疫苗（pcDNA3.1-pp65）纯化工艺及质量控制标准,为进一步研究奠定了重要基础。     

2项对诊断儿童人巨细胞病毒活动性感染的比较

目的比较血清人巨细胞病毒(HCMV)-IgM抗体(以下简称IgM抗体)与HCMV pp65抗原(以下简称pp65)两种方法对HCMV活动性感染诊断检测的实用意义。方法采集临床疑似HMCV活动性感染儿童血标本91份,分离血浆和多形白细胞,分别用于IgM抗体检测和pp65抗原检测,同时用聚合酶链反应(PCR)检测HCMV DNA,与pp65抗原作平行比较。结果 pp65抗原检测的结果与IgM抗体检测的符合率为70.3%,与HC-MV DNA检测相比,pp65抗原检测的符合率、特异性和敏感度分别为85.5%、70.6%和75.6%,而且高pp65抗原血症与患者临床症状密切相关。结论 pp65抗原血症反映该病毒活动状况,可监测HCMV活动性感染。     

Visualization of the dynamic multimerization of human Cytomegalovirus pp65 in punctuate nuclear foci

The phosphorylated protein pp65 of human Cytomegalovirus (HCMV) is the predominant virion protein and the major tegument constituent. It plays important roles in HCMV infection and virion assembly. Live cell imaging and fluorescence recovery after photobleaching (FRAP) analysis showed that HCMV pp65 accumulated dynamically in punctuate nuclear foci when transiently expressed in mammalian cells. Fluorescence resonance energy transfer (FRET) imaging disclosed that pp65 can self-interact in its localization foci. Yeast two-hybrid assay verified that pp65 is a self-associating protein, and the N-terminal amino acids 14–22 were determined to be essential for pp65 self-association. However, these amino acids were not related to pp65 localization in the specific nuclear foci. The interaction of pp65 and ppUL97 was also studied by FRET microscopy, and the result suggested that there is another signal sequence in pp65, being the ppUL97 phosphorylation site, that is responsible for localization of pp65 in nuclear foci. These results help to understand the function of pp65 in HCMV infection and virion morphogenesis.     

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.