A novel virus-encoded nucleocytoplasmic shuttling protein: the UL3 protein of herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) UL3 protein is a nuclear protein. In this study, the molecular mechanism of the subcellular localization of UL3 was characterized by fluorescence microscopy in living cells. A nuclear localization signal (NLS) and a nuclear export signal (NES) were also identified. UL3 was demonstrated to target to the cytoplasm through the NES via chromosomal region maintenance 1 (CRM-1) dependent pathway, and to the nucleus through RanGTP-dependent mechanism. Heterokaryon assays confirmed that UL3 was capable of shuttling between the nucleus and the cytoplasm. These results demonstrate that the UL3 protein is a novel HSV-1 encoded nucleocytoplasmic shuttling protein.     

IgG antibodies to human cytomegalovirus late protein UL94 in patients with systemic sclerosis

Human cytomegalovirus (HCMV) has been proposed as an amplifying agent for at least some of the spectrum of systemic sclerosis (SSc; scleroderma). In support of this hypothesis, antibodies to the HCMV late protein UL94 have been detected in the majority of SSc patients in a study involving Caucasian subjects from Italy. The aim of this investigation was to determine whether elevated levels of anti-UL94 antibodies are present in African American and Caucasian SSc patients from the U.S. We further wished to determine whether there was a significant difference in the levels of anti-UL94 antibodies between the diffuse and the limited forms of the disease. IgG antibodies to a UL94 peptide were measured in 254 Caucasian and 90 African American subjects by an enzyme-linked immunosorbent assay (ELISA). In both Caucasian and African American subjects, the mean antibody level in the diffuse form of SSc was significantly higher than that in the respective control subjects (714 vs. 466 ng/ml, p = 0.005; 1226 vs. 512 ng/ml, p < 0.0001). Also, among Caucasian SSc patients, the mean antibody level in the diffuse form of SSc was significantly higher than that in the limited form of the disease (714 vs. 465 ng/ml, p = 0.02). These results show that increased levels of antibodies to the HCMV late protein UL94 are associated with SSc and they may be a marker for the severity of the disease.     

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.     

人巨细胞病毒基质蛋白PP150是CTL识别的提呈抗原之一

用痘苗病毒载体ＰＳＣ１１构建编码人巨细胞病毒（ＣＭＶ）基质蛋白ＰＰ１５０的重组痘苗病毒，刺激５例血清ＣＭＶ阳性供体全部诱导出ＰＰ１５０特异性细胞毒性Ｔ淋巴细胞（ＣＴＬ）反应。ＰＰ１５０特异性ＣＴＬ不仅能溶解Ｖａｃ．ＰＰ１５０感染细胞，也溶解ＣＭＶ感染细胞。在病毒感染早期（２小时）和用ＲＮＡ合成抑制剂ＡｃｔＤ阻止感染细胞病毒基因转录的条件下，ＰＰ１５０特异性ＣＴＬ能同样有效地溶解ＣＭＶ感染细胞。提示随病毒颗粒渗入的ＰＰ１５０在ＣＭＶＤＮＡ复制前即能有效地被提呈。上述结果表明，ＰＰ１５０是ＣＴＬ识别的抗原之一。     

Human cytomegalovirus UL131A, UL130 and UL128 genes are highly conserved among field isolates

Summary. Coding sequences of the UL131A, UL130, and UL128 genes of human cytomegalovirus (HCMV) were found to be highly conserved among 34 field isolates from pregnant women with primary HCMV infection and their fetuses or newborns, as well as from solid organ transplant recipients and patients with AIDS. No strain clustering was observed. In contrast, sequencing of UL55 (gB coding gene) allowed the 34 isolates to be clustered into 4 genotypes. The conservation of the UL131A-UL128 locus is consistent with the conclusion that the three encoded proteins are all essential for growth of HCMV in endothelial cells and virus transfer to leukocytes.     

Human cytomegalovirus a sequence and UL144 variability in strains from infected children

Human cytomegalovirus (HCMV) displays genetic polymorphisms. This variability may contribute to strain-specific tissue tropism and disease expression in HCMV-infected humans. To determine strain variability in a sequence and UL144 gene regions, 51 low-passage isolates from 44 HCMV-infected children were studied. Isolates were obtained from 28 healthy children attending child care centers in Iowa and from 16 congenitally infected infants born in Texas. Isolates demonstrated substantial nucleotide variation in each gene region. Phylogenetic analysis of a sequence variability allowed 39 isolates to be grouped into six clades. The largest clade contained 16 isolates with > or = 95% nucleotide homology. Forty-eight of the 49 HCMV isolates yielding UL144 amplicons was grouped according to the clades described a few years ago [Lurain et al. (1999) Journal of Virology 73:10040-10050]. No linkage was observed among a sequence, UL144, and glycoprotein B (gB; UL55) polymorphisms. Four Texas and 11 Iowa isolates displayed > or = 95% sequence homology for a sequence and UL144 regions and possessed identical gB genotypes. No relationship between UL144 polymorphisms and outcome of congenital HCMV infection was observed. These data indicate that HCMV strains circulating among young children have UL144 polymorphisms similar to those of HCMV strains excreted by immunocompromised adults. Identification of conserved nucleotide sequences among Iowa and Texas children suggests genetic stability and biologic importance of these gene regions.     

Antibodies against human cytomegalovirus late protein UL94 in the pathogenesis of scleroderma-like skin lesions in chronic graft-versus-host disease

Human cytomegalovirus (hCMV) infection and its reactivation correlate both with the increased risk and with the worsening of graft-versus-host disease (GVHD). Because scleroderma-like skin lesions can occur in chronic GVHD (cGVHD) in allogeneic stem-cell transplant (HCT) patients and hCMV is relevant in the pathogenesis of systemic sclerosis (SSc), we evaluated the possible pathogenetic link between hCMV and skin cGVHD. Plasma from 18 HCT patients was tested for anti-UL94 and/or anti-NAG-2 antibodies, identified in SSc patients, by direct ELISA assays. Both donors and recipients were anti-hCMV IgG positive, without autoimmune diseases. Patients' purified anti-UL94 and anti-NAG-2 IgG binding to human umbilical endothelial cells (HUVECs) and fibroblasts was performed by FACS analysis and ELISA test. HUVECs apoptosis and fibroblasts proliferation induced by patients' anti-NAG-2 antibodies were measured by DNA fragmentation and cell viability, respectively. About 11/18 patients developed cGVHD and all of them showed skin involvement, ranging from diffuse SSc-like lesions to limited erythema. Eight of eleven cGVHD patients were positive for anti-UL94 and/or anti-NAG-2 antibodies. Remarkably, 4/5 patients who developed diffuse or limited SSc-like lesions had antibodies directed against both UL94 and NAG-2; their anti-NAG-2 IgG-bound HUVECs and fibroblasts induce both endothelial cell apoptosis and fibroblasts proliferation, similar to that induced by purified anti-UL94 and anti-NAG-2 antibodies obtained from SSc patients. In conclusion, our data suggest a pathogenetic link between hCMV infection and scleroderma-like skin cGVHD in HCT patients through a mechanism of molecular mimicry between UL94 viral protein and NAG-2 molecule, as observed in patients with SSc.     

Human cytomegalovirus open reading frame UL11 encodes a highly polymorphic protein expressed on the infected cell surface

Summary.  Human cytomegalovirus (HCMV) open reading frame (ORF) UL11 locates within a polymorphic region of the viral genome identified previously by a restriction-fragment-length-polymorphism. We report here that ORF UL11 encodes a polymorphic protein expressed on the surface of HCMV-infected cells. First, we determined the nucleotide sequence of ORF UL11 from ten strains and compared it among the strains. Out of 205 amino acids consisting of the predicted N-terminal region beside the putative transmembrane stretch in strain AD169, 88 residues were divergent on more than one strain. In contrast, the predicted C-terminal side including the putative transmembrane domain was identical at the amino acid sequence level. In addition, the number and location of predicted cysteine residue were also conserved. Next, we screened a cDNA library from HCMV-infected cells and obtained a cDNA clone containing the full-length ORF UL11. Finally, we identified the gene product of UL11 on the surface of HCMV-infected cells by FACS analysis with polyclonal antibodies generated against a glutathione S-transferase/UL11 fusion protein. The fusion protein contained a region within the N-terminal side next to the predicted transmembrane stretch. These results indicate that the N-terminal side of UL11 protein containing variable amino acid residues protrudes from the infected cell surface. Accepted January 27, 1997; Received November 7, 1996     

Ganciclovir resistance mutations in UL97 and UL54 genes of Human cytomegalovirus isolates resistant to ganciclovir

Human cytomegalovirus (HCMV) resistance to ganciclovir results from mutations in viral phosphotransferase (UL97) and/or DNA polymerase (UL54) genes. The HCMV isolates from the blood of immunocompromised patients with persisting presence of the pp65 antigen in the blood in spite of ganciclovir therapy were tested for ganciclovir susceptibility by an immediate-early antigen plaque reduction assay, and the UL54 and UL97 genes were sequenced. Nine isolates from eight patients (six patients with acquired immune deficiency syndrome (AIDS), one liver transplant recipient and one renal transplant recipient) showed phenotypic resistance to ganciclovir. All these ganciclovir-resistant HCMV isolates harbored one or more of the following UL97 mutations: M460V, A594V, A594T, L595S, C603W, and M615V. Two isolates harbored the P522S mutation in the UL54 gene. The M615V mutation in the UL97 gene has not been reported earlier and its role in ganciclovir resistance remains to be elucidated. In ganciclovir-resistant HCMV isolates the UL54 gene was less frequently mutated than the UL97 gene. The P522S mutation was relatively frequent in UL54-mutated HCMV isolates.     

Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase

Summary. The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy. Received September 2, 1997 Accepted January 14, 1998     

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.     

Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35

The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.     

干扰素诱导蛋白Nmi与人巨细胞病毒皮层蛋白UL23相互作用区域的研究

目的:探究干扰素诱导蛋白N-Myc相互作用因子(Nmi)与人巨细胞病毒(HCMV)皮层蛋白UL23相互作用的关键区域。方法:根据前期实验结果,分别将10个不同截短突变型的Nmi构建到原核表达载体p GEX-4T-1上,转化到大肠杆菌Rosetta(DE3)感受态细胞中,表达并纯化出带有GST标签的融合蛋白,利用GST-pulldown的方法探究Nmi与UL23相互作用的区域。根据GST-Pulldown实验结果,用同源重组的方法在真核表达载体pc DNA4-Myc上分别构建3个缺失突变型Nmi。将实验组和对照组的Nmi分别与含有Flag标签的UL23质粒共转染至He La细胞中,通过免疫共沉淀法进一步研究Nmi与UL23的相互作用区域。结果:(1)10个截短突变型Nmi与GST基因融合的原核表达载体构建成功;(2)3个缺失突变型Nmi与Myc基因融合的真核表达载体构建成功;(3)GST-pulldown实验证明Nmi与UL23相互作用位点位于Nmi上的第192~202位氨基酸区域;(4)免疫共沉淀法确认Nmi的第192~202位氨基酸区域是与UL23相互作用的区域,与GST-pulldown实验结果一致。结论:Nmi与UL23相互作用的区域位于Nmi上第192~202位氨基酸区域。这为阐明UL23帮助HCMV在宿主体内潜伏的分子机理提供了基础。