Interactions between human cytomegalovirus helicase-primase proteins

The human cytomegalovirus (HCMV) UL70, UL102, and UL105 genes are predicted to encode essential proteins that assemble the replicative helicase-primase complex based on sequence and genome position similarities to putative herpes simplex virus type 1 (HSV-1) counterparts. Consistent with this prediction, they are required for transient complementation of DNA synthesis. However, little is known about their physical interactions and biochemical activities, primarily because of their restricted expression in HCMV-infected cells. To look for assembly of the predicted complexes, we prepared rabbit polyclonal antisera and used Semliki Forest Virus (SFV) vectors to express untagged and glutathione-S-transferase (GST)-tagged UL70, UL102 and UL105 proteins. The UL70 and UL105 proteins co-purified with the GST-tagged UL102 protein from triply-infected baby hamster kidney cells (BHK-21), and pUL70, but not pUL105, co-purified with pGST-UL102 from dually infected BHK-21 cells. In immunoprecipitation experiments with untagged SFV-expressed proteins, pUL70 or pUL105 coprecipitated with pUL102, pUL102 or pUL70 co-precipitated with pUL105; and pUL102 or pUL105 coprecipitated with pUL70. Comparison of the GST-pull down and immunoprecipitation experiments suggested that the amino-terminal GST-tag interfered with certain pairwise interactions. These results support the prediction that the HCMV helicase-primase proteins assemble a three-protein heteromeric complex, and show that each protein contacts both partners.     

ATPase 抑制因子1是与HCMV pUL23蛋白相作用的宿主蛋白分子-酵母双杂交技术筛选与鉴定

目的： 利用酵母双杂交技术筛选与人巨细胞病毒相互作用的宿主蛋白分子,为探讨人巨细胞病毒pUL23蛋白在HCMV生活周期中的作用机制提供依据。方法: 利用GAL4酵母双杂交系统筛选人胚肾cDNA文库,以获得与人巨细胞病毒pUL23蛋白相互作用的宿主蛋白分子,再通过回交试验和体外GST-pulldown试验验证两者之间的相互作用。结果: 酵母双杂交筛选得到宿主蛋白分子ATPase inhibitory factor 1（ATIF1）,回交试验和体外GST-pulldown试验再次确认ATIF1能够与人巨细胞病毒pUL23蛋白相互作用。结论: pUL23确实能够与ATIF1相互作用,它们之间的相互作用可能为研究pUL23在病毒生活周期发挥的功能提供依据。     

Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art

Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. The significant clinical impact of HCMV infection and progression to HCMV disease among allogeneic hematopoietic stem cell transplant recipients has been reduced by prophylactic, preemptive, and curative treatments using ganciclovir, valganciclovir, foscarnet, and cidofovir. Resistance to (val)ganciclovir results from mutations localized in HCMV UL97 gene (encoding the pUL97 phosphotransferase), UL54 gene (encoding the pUL54 DNA polymerase), or both genes, whereas foscarnet and cidofovir resistance results from mutations localized within UL54 gene only. This review is focused on HCMV antiviral drug resistance, including the functions of target genes of antivirals, the mechanisms of antiviral resistance, the different mutations in pUL97 and pUL54 that have been identified in either clinical isolates or laboratory strains, and their impact on HCMV susceptibility to antiviral drugs. It emphasizes the importance of proving that observed genetic changes confer resistance so they can be distinguished from polymorphisms. Because of the emergence of HCMV resistance to currently available drugs, novel drugs are urgently needed for the therapeutic management of HCMV‐resistant infections in hematopoietic stem cell transplant patients. Copyright © 2016 John Wiley & Sons, Ltd.     

The product of human cytomegalovirus UL73 is a new polymorphic structural glycoprotein (gpUL73)

OBJECTIVES: This work focuses on human cytomegalovirus (HCMV) UL73, which encodes for a putative transmembrane glycoprotein that is highly conserved among herpesviruses. STUDY DESIGN: pUL73 expression was analyzed both in transiently transfected and in HCMV-infected cells using a pUL73-specific antiserum by immunoblot and immunofluorescence. Sequencing analysis from several clinical isolates and laboratory-adapted strains was also performed. RESULTS: pUL73 expressed in transiently transfected cells consists in a polypeptide of the expected size (15-18 kd) with cytoplasmic localization. In infected cells, pUL73 is expressed with true-late kinetics and localizes both in perinuclear granular formations and on the cell surface. A broad band (39-53 kd), sensitive to O-glycosidase digestion was detected in purified virus. In addition, sequence analysis showed that the N-terminal portion of pUL73 from clinical isolates is highly polymorphic. CONCLUSIONS: UL73 encodes for a new structural glycoprotein (gpUL73) expressed on the cell surface of infected cells and highly polymorphic among clinical isolates.     

稳定表达人巨细胞病毒蛋白pUL23细胞系建立及其功能研究

目的:建立稳定表达人巨细胞病毒UL23基因的HELF细胞系,研究病毒蛋白在宿主细胞内行为,为进一步研究人巨细胞病毒蛋白pUL23的功能提供依据。方法:通过PCR技术从人巨细胞病毒基因组中扩增出UL23基因,通过分子克隆技术构建重组逆转录病毒表达载体pLEGFP-N1-FLAG-UL23。将该载体导入Am-phoPackTM-293细胞,收获重组逆转录病毒,然后感染HELF细胞,HELF经持续G418抗性筛选后获得稳定表达UL23基因的细胞系。采用激光共聚焦显微镜观察病毒蛋白在细胞内的定位。结果:RT-PCR、Western blotting结果证实病毒基因UL23能够整合到宿主细胞基因组中,并能在宿主细胞中稳定表达病毒蛋白。共聚焦显微镜观察到病毒蛋白pUL23定位于细胞质,处于细胞核周边。结论:利用逆转录病毒载体介导的基因转移技术,成功构建了稳定表达UL23基因的转基因细胞系。该病毒蛋白在宿主细胞质中的定位,提示病毒蛋白发挥功能的空间位于细胞核周边,有利地推进了人巨细胞病毒蛋白pUL23功能研究的进程。     

Characterization of the guinea pig cytomegalovirus genome locus that encodes homologs of human cytomegalovirus major immediate-early genes, UL128, and UL130

We reported previously that the guinea pig cytomegalovirus (CMV) stock purchased from the American Type Culture Collection contained two types of strains, one containing and the other lacking a 1.6 kb locus, and that the 1.6 kb locus was required for efficient viral growth in animals but not in cell culture. In this study, we characterized the genetic contents of the locus, and found that i) the 1.6 kb locus encodes homologs of human CMV UL128 and UL130, GP129 and GP131, respectively, ii) these genes are expressed with late gene kinetics, iii) GP131 protein (pGP131) localized to cell surface only in the presence of glycoproteins H and L, and iv) pGP131 is a virion component. Therefore, it is plausible that pGP131 forms a complex with glycoproteins H and L and becomes a virion component as does UL130 protein (pUL130). Since pUL130 is one of the glycoproteins essential for infection of endothelial and epithelial cells in human and primates, functional and immunological analyses of this GPCMV homolog of pUL130 may help to illuminate the in vivo role of pUL130.     

Evidence that the human cytomegalovirus 46-kDa UL72 protein is not an active dUTPase but a late protein dispensable for replication in fibroblasts

The Human Cytomegalovirus (HCMV) UL72 gene is considered to be the equivalent of the dUTPase gene of the Alpha- and Gamma-herpesviruses. To characterize its function, the expression profiles of UL72 at both the RNA and the protein level were determined. The gene is expressed with a late kinetics and the corresponding UL72 46-kDa protein accumulates late during infection in the cytoplasm of infected cells. The pUL72 was expressed in E. coli and the purified recombinant protein did not display a detectable dUTPase activity. The viral yields of reconstituted HCMV RVDeltaUL72 viruses carrying a deletion within the UL72 ORF demonstrated a moderate growth defect following low MOI infections, whereas their DNA synthesis profiles were not significantly different from those of the parental HCMV RVAD169. These results demonstrate that the UL72 gene product is not a dUTPase and is not essential for replication in human fibroblasts.     

The tegument protein UL94 of human cytomegalovirus as a binding partner for tegument protein pp28 identified by intracellular imaging

The tegument protein pp28 of human cytomegalovirus (HCMV) is essential for the assembly of infectious HCMV virions, but how it functions during the process of HCMV tegumentation and envelopment remains unclear. By using live cell fluorescence resonance energy transfer (FRET) microscopy and yeast two-hybrid assays, we found that another HCMV tegument protein, UL94, was a specific binding partner for pp28. The interaction between pp28 and UL94 was imaged in a punctuate, juxtanuclear compartment, previously designated as the virus assembly compartment (AC). Amino acids 22-43 of pp28 were identified as being responsible for its binding with UL94, while no linear binding site could be found within UL94. The interaction between pp28 and UL94 may serve as a link in the sequential processes of HCMV capsidation, tegumentation and envelopment. This study provides a foundation for further studies into how the HCMV tegument proteins act in the assembly of HCMV virions.     

Efficient inhibition of human cytomegalovirus UL122 gene expression in cell by small interfering RNAs

In order to develop a gene therapy to human cytomegalovirus (HCMV), RNA interference (RNAi) was employed to inhibit the expression of HCMV UL122 gene in vitro. Recombinant vector pUL122‐EGFP, which expressed UL122‐EGFP fusion protein, and recombinant vectors psi122‐1, psi122‐2 and psi122‐3, which expressed small interfering RNAs (siRNAs) targeted to UL122 were contransfected into AD293 cells. The fluorescence signal of pUL122‐EGFP was greatly suppressed by psi122‐1 and psi122‐2, with an inhibitory rate of 82.0% ± 1.0% and 79.5% ± 2.5%, respectively. The mRNA of pUL122‐EGFP of the cells transfected with psi122‐1 and psi122‐2 was decreased 97.3% ± 0.6% and 98.0% ± 0.1%, respectively. Vector psi122‐3 showed a slightly low suppression rate. Therefore, it may be concluded that plasmids encoding siRNAs targeted to UL122 is able to in vitro reduce markedly the expression of UL122‐EGFP. And it is very likely that the psi122‐1 and psi122‐2 are potentially efficacious siRNAs in the gene therapy of HCMV infection in vivo, in which further investigations are required. This study is expected to greatly facilitate the use of the RNAi technology for the anti‐HCMV studies. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular dissection of the human B cell response against cytomegalovirus infection by lambda display

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, is the main cause of congenital abnormalities and mental retardation in newborns and is also responsible for severe life-threatening complications in immunocompromised individuals, including AIDS patients and transplant recipients. The disorders generated by cytomegalovirus are closely associated with the competence of the host immune system and both humoral and cell-mediated mechanisms are involved in the response to viral infection. To identify viral proteins recognized by host antibody responses, a cytomegalovirus genome library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals with congenital or acquired infection allowed the identification of a wide panel of recombinant bacteriophages carrying cytomegalovirus B cell epitopes. Epitope-containing fragments within the families of tegument proteins (pUL25, pUL32), structural proteins (pUL48, pUL56) and glycoproteins (pUL55) were identified. Moreover, library screening permitted isolation of phage clones carrying an antigenic region of an uncharacterized HCMV protein encoded by the UL71 open reading frame (ORF), highlighting the potential of lambda display technology in antigen and epitope discovery.     

Distribution of UL144, US28 and UL55 genotypes in Polish newborns with congenital cytomegalovirus infections

Human cytomegalovirus (HCMV) is the most common congenital infection. HCMV strains display genetic variability in different regions. Distribution of HCMV genotypes in the population of congenitally infected newborns from Central Poland and viral load in newborns' blood is described and discussed. HCMV isolates were analysed by sequencing at three sites on the genome: the UL144 tumour necrosis factor-alpha (TNFα)-like receptor gene, the US28 beta-chemokine receptor gene and the UL55 envelope glycoprotein B (gB) gene. The newborns' blood was examined for HCMV DNA with a nested (UL144, UL55) or heminested (US28) polymerase chain reaction, and the genotypes were determined by sequence analysis. HCMV DNA was detectable in 25 out of 55 examined newborns born by HCMV-infected mothers (45.5%). The blood viral load in mother-infant pairs was determined. Most of the newborns had identical virus genotype, gB2 (96%), UL144 B1 (88%) and US28 A2 (84%). These genotypes were detected in all newborns with asymptomatic congenital infection. The occurrence of UL144 B1 or US28 A2 genotypes in the babies examined was significant in comparison to other genotypes (p=0.0002 and p=0.040 respectively). There was no association between specific gB subtypes in all patients groups (p=0.463). There was no correlation between HCMV genotypes and the outcome.     

Consequences of human cytomegalovirus mimicry

The HCMV genome has evolved with its host by incorporating a series of genes that are homologous to, or functionally mimic, cellular genes. Some are designed to counteract the stress of infection on the host cell, notably the viral antiapoptotic proteins (vICA, vMIA). Others potentially help the infected cell maintain a low immunologic profile. These include virus-encoded chemokine receptors (UL33, UL78, US27, US28), FcRs (gp TRL11/IRL11, gp UL119-118), and proteins that directly or indirectly thwart natural killer cell activity (UL16, gpUL40). In addition, some viral proteins may play a role in immunopathology because of fortuitous cross-reactivity with host cell proteins. This overview discusses how these proteins affect the life of the host cell and its immediate neighbors.     

Human cytomegalovirus UL94 is a nucleocytoplasmic shuttling protein containing two NLSs and one NES

The tegument protein UL94 is a human cytomegalovirus (HCMV) late protein and its function has yet to be determined. Using live cell fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) imaging, we found that UL94 is able to shuttle between the nucleus and cytoplasm. Analysis of UL94 mutants fused to EGFP showed that two newly characterized nuclear localization sequences (NLSs) and amino acid 343 play key roles in UL94 nuclear localization. Mutation of these sequences can alter the intracellular distribution of UL94 and disrupt its nucleocytoplasmic shuttling. Amino acid 343 of UL94 was also found to be crucial for its interaction with another HCMV tegument protein pp28. Furthermore, one nuclear export sequence (NES) was identified within UL94. Mutation of the key amino acids in the NES can also alter the intracellular distribution of UL94 and disrupt its shuttling function. Like other proteins containing a leucine-rich export signal, nuclear export of the UL94 was affected by leptomycin B, indicating that it is exported via the Crm1-dependent pathway. Our data provide a basis for further understanding the character and function of HCMV UL94.     

Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein

Human cytomegalovirus (HCMV) has evolved a multitude of molecular mechanisms to evade the antiviral immune defense of the host. Recently, using soluble recombinant molecules, the HCMV UL16 glycoprotein was shown to interact with some ligands of the activating immunoreceptor NKG2D and, therefore, may also function as a viral immunomodulator. However, the role of UL16 during the course of HCMV infection remained unclear. Here, we demonstrate that HCMV infection of fibroblasts induces expression of all known NKG2D ligands (NKG2DL). However, solely MICA and ULBP3 reach the cellular surface to engage NKG2D, whereas MICB, ULBP1 and ULBP2 are selectively retained in the endoplasmic reticulum by UL16. UL16-mediated reduction of NKG2DL cell surface density diminished NK cytotoxicity. Thus, UL16 functions by capturing activating ligands for cytotoxic lymphocytes that are synthesized in response to HCMV infection.     

Identification and functional analysis of the small membrane-associated protein pUL11 of avian infectious laryngotracheitis virus

pUL11 is a highly conserved, small, acylated, membrane-associated tegument protein of herpesviruses. It is involved in final envelopment of nascent virions in the cytoplasm, although the precise mechanism is still unknown. By screening of mouse monoclonal antibodies (mAb) raised against purified particles of infectious laryngotracheitis virus (ILTV) of chickens (Veits et al., 2003a), we identified two mAb recognizing the 15 kDa UL11 protein (pUL11) of this avian alphaherpesvirus. These mAb permitted detection and precise localization of pUL11 in mature ILT virions, as well as in the cytoplasm of infected chicken cells by Western blot analyses, indirect immunofluorescence tests, and immunoelectron microscopy. For investigation of gene function UL11-deleted ILTV recombinants were generated. Like its homologues in several other alphaherpesviruses, ILTV-pUL11 was shown to be nonessential for productive virus replication. However, compared to wild-type and UL11 rescued ILTV the deletion mutants exhibited significantly reduced virus yields and moderately impaired spread in cell culture. In the absence of pUL11, electron microscopy of infected cells revealed accumulations of tegument proteins with nucleocapsids, and marked distortions of Golgi membranes in the cytoplasm, which obviously inhibited the formation of mature, enveloped virus particles. Taken together, our results demonstrate that pUL11 is relevant for secondary envelopment of ILTV, and confirm functional conservation of this protein in herpesviruses. The now available unique pUL11-specific mAb will help to further analyze this function, which is presumably mediated by physical interactions with other viral gene products, in cultured cells and in the natural animal host of ILTV.