Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS.

Three human cytomegalovirus (HCMV) strains (VR4760, VR4955, and VR5120) showing double resistance to ganciclovir (GCV) and foscarnet (PFA) were isolated from three patients with AIDS who underwent multiple sequential courses of therapy with GCV and PFA (A. Sarasini, F. Baldanti, M. Furione, E. Percivalle, R. Brerra, M. Barbi, and G. Gerna, J. Med. Virol., 47:237-244, 1995). We previously demonstrated that the three strains were genetically unrelated and that each of them was present as a single viral population in vivo. Thus, in each of the three cases, a single viral strain was resistant to both GCV and PFA. In the present paper, we report the characterization of the molecular bases of the double resistance and demonstrate that the PFA resistance is associated with a slower replication of HCMV strains in cell cultures. Sequencing of the UL97 and UL54 genes, GCV anabolism assays, and marker transfer experiments showed that GCV resistance was due to single amino acid changes in the UL97 gene product (VR4760, Met-460 --> Ile; VR4955, Ala-594 --> Val; VR5120, Leu595 --> Ser), while single amino acid changes in domain II of the DNA polymerase (VR4760 and VR5120, Val-715 --> Met; VR4955, Thr-700 --> Ala) were responsible for both the PFA resistance and the slow-growth phenotype. Thus, in these three cases, double resistance to GCV and PFA was not due to a single mutation conferring cross-resistance or to the presence of a mixture of strains with different drug susceptibilities. The HCMV DNA polymerase recombinant strains carrying the mutations conferring PFA resistance were sensitive to GCV and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). In addition, the same UL54 mutations were responsible for the slow growth of the clinical isolates, since the recombinant strains showed a marked delay in immediate-early antigen plaque formation and a reduction of infectious virus yield compared with AD169, from which they were derived. These results may have some important implications for the successful isolation, propagation, and characterization of PFA-resistant strains from clinical samples containing mixed viral populations.     

Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir.

The same point mutation in the human cytomegalovirus UL97 open reading frame was found in three independently isolated ganciclovir-resistant mutants of strain AD169. Point mutations in the DNA polymerase genes of these strains have been previously identified (N.S. Lurain, K.D. Thompson, E.W. Holmes, and G.S. Read, J. Virol. 66:7146-7152, 1992). All three strains are, therefore, double mutants. To determine the contribution of the UL97 mutation to the high ganciclovir resistance of these mutants, the mutation from the ganciclovir-resistant strain D6/3/1 was transferred to the wild-type strain AD169 to produce the recombinant R6HS. The ganciclovir resistance of R6HS is 4-fold lower than that of D6/3/1 but 10-fold higher than that of AD169. R6HS, like AD169, is sensitive to the nucleotide analogs (S)-1-[(3-hydroxy-2-phosphonylmethoxy) propyl]adenine and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine. Ganciclovir phosphorylation in R6HS-infected cells was at the same reduced level as that found in cells infected with the parental mutant D6/3/1. The same G-to-T transversion at nucleotide 1380 in the UL97 coding sequence is present in both R6HS and D6/3/1. This mutation results in the substitution of isoleucine for methionine at amino acid residue 460. In an alignment of the R6HS UL97 amino acid sequence with the amino acid sequences of a wide range of protein kinase family members, methionine 460 lies within a highly conserved region which may function in nucleotide binding and phosphate transfer.     

Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism

Epithelial cells are one of the prominent cell types infected by human cytomegalovirus (HCMV) within its host. However, many cultured epithelial cells, such as ARPE-19 retinal pigmented epithelial cells, are poorly infected by laboratory-adapted strains in cell culture, and little is known about the viral factors that determine HCMV epithelial cell tropism. In this report, we demonstrate that the UL131 open reading frame (ORF), and likely the entire UL131-128 locus, is required for efficient infection of epithelial cells. Repair of the mutated UL131 gene in the AD169 laboratory strain of HCMV restored its ability to infect both epithelial and endothelial cells while compromising its ability to replicate in fibroblasts. ARPE-19 epithelial cells support replication of the repaired AD169 virus as well as clinical isolates of HCMV. Productive infection of cultured epithelial cells, endothelial cells, and fibroblasts with the repaired AD169 virus leads to extensive membrane fusion and syncytium formation, suggesting that the virus may spread through cell-cell fusion.     

Resistance of human cytomegalovirus to the benzimidazole L-ribonucleoside maribavir maps to UL27

1-(beta-D-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(beta-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.     

The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase.

The temporal expression of the UL97 gene product during human cytomegalovirus (HCMV) infection of human foreskin fibroblasts (HFF) and subcellular localization of this protein were analyzed by using a polyclonal antiserum raised against a truncated UL97 protein of 47 kDa. The UL97 protein was detectable 16 h after infection by Western blot (immunoblot) analysis. Since only reduced UL97 expression occurred in the presence of two inhibitors of DNA replication, phosphonoacetic acid and ganciclovir, we conclude that UL97 is an early-late gene, requiring DNA replication for maximum expression. By indirect immunofluorescence, the protein could be visualized in the nuclei of virus-infected HFF 22 h after infection. Nuclear localization of the UL97 protein was also detected in thymidine kinase-deficient 143B cells infected with a recombinant vaccinia virus containing the entire UL97 open reading frame (ORF), as well as in HFF transiently expressing the entire UL97 ORF under the control of HCMV major immediate-early promoter. However, transiently expressed 5'-terminal deletion mutants of the UL97 ORF in addition showed a cytoplasmic localization of the UL97 protein, confirming the presence of a nuclear localization site in the N-terminal region of the protein. Our high-pressure liquid chromatography analyses confirmed the ganciclovir phosphorylation by the UL97 protein, but no specific phosphorylation of natural nucleosides was observed, indicating that the UL97 protein is not a nucleoside kinase. During plaque purification of recombinant UL97-deficient HCMV, this virus was growth defective; hence, we presume that UL97 may be essential for the viral life cycle.     

The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host

The human herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), HHV-6B, HHV-7 and HHV-8, establish persistent infections with possible recurrence during immunosuppression. HCMV replication is inhibited by the nucleoside analogue ganciclovir (GCV), the compound of choice for the treatment of HCMV diseases and preemptive treatment of infections. The viral UL97 protein (pUL97) which shares homologies with protein kinases and bacterial phosphotransferases is able to monophosphorylate GCV. Homologues of pUL97 are found in HSV (UL13), VZV (ORF47), EBV (BGLF4), HHV-6 (U69), HHV-8 (ORF36) as well as in murine CMV (M97) or rat CMV (R97). Several indolocarbazoles have been reported to be specific inhibitors of pUL97. The protein is important for efficient replication of the virus. Autophosphorylation of pUL97 was observed using different experimental systems. Most recently, it has been shown that pUL97 interacts with the DNA polymerase processivity factor pUL44. Indolocarbazole protein kinase inhibitors are promising lead compounds for the development of more specific inhibitors of HCMV.     

人类巨细胞病毒UL133基因在临床低传代株中的多态性研究

人类巨细胞病毒在多次传代后,会表现出不同的毒力水平.与临床低传代株Toledo相比,实验室高传代株AD169缺失了19个开放阅读框(ORF).这19个基因被认为是与HCMV致病性最可能相关的一组基因,研究这些基因的多态性对揭示HCMV致病性的遗传基础具有指导意义.UL133基因是这19个ORF中的一个.以临床低传代株Toledo和Merlin为对照,分析了23个临床病毒株UL133基因的遗传多态性.序列分析表明,UL133基因具有一定的多态性,Toledo株、Merlin株与我们分离到的临床株一起可分为3个基因型:G1、G2和G3.G2、G3型毒株均能导致先天性感染.没有发现UL133基因型与患儿临床疾病的必然关联.     

Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus.

We analyzed whether the phosphotransferase encoded by the UL97 open reading frame of human cytomegalovirus (HCMV) alone is sufficient to confer ganciclovir (GCV) susceptibility to a foreign virus. Two vaccinia virus recombinants (T1 and A5) containing the UL97 open reading frames from a GCV-sensitive HCMV and from a GCV-resistant strain were constructed. T1 exhibited a GCV-sensitive phenotype in plaque reduction assays, whereas A5 did not. Moreover, T1-infected cell cultures showed a strongly increased incorporation of [14C]GCV triphosphate into macromolecular DNA, compared with recombinant A5 or vaccinia virus controls, which could be inhibited by the addition of guanosine. This shows that UL97 kinase is the only additional gene product required to make vaccinia virus susceptible to GCV, and guanosine seems to be one natural substrate for the enzyme. The system described here should be very helpful for fast and detailed functional analyses of UL97 mutations found in GCV-resistant HCMV isolates.     

Characterization of Drug Resistance-Associated Mutations in the Human Cytomegalovirus DNA Polymerase Gene by Using Recombinant Mutant Viruses Generated from Overlapping DNA Fragments

A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts, and their drug susceptibility profiles were determined. Amino acid substitutions located in UL54 conserved region IV (N408D, F412C, and F412V), region V (A987G), and δ-region C (L501I, K513E, P522S, and L545S) conferred various levels of resistance to cidofovir and ganciclovir. Mutations in region II (T700A and V715M) and region VI (V781I) were associated with resistance to foscarnet and adefovir. The region II mutations also conferred moderate resistance to lobucavir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region III (L802M, K805Q, and T821I) were associated with various drug susceptibility profiles. Mutations located outside the known UL54 conserved regions (S676G and V759M) did not confer any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations with respect to the final drug resistance phenotype was demonstrated. Finally, the influence of selected UL54 mutations on the susceptibility of viral DNA replication to antiviral drugs was characterized by using a transient-transfection-plus-infection assay. Results of this work exemplify specific roles of the UL54 conserved regions in the development of HCMV drug resistance and may help guide optimization of HCMV therapy.     

Comparison between human cytomegalovirus pUL97 and murine cytomegalovirus (MCMV) pM97 expressed by MCMV and vaccinia virus: pM97 does not confer ganciclovir sensitivity

The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056-7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.     

Human cytomegalovirus UL97 kinase and nonkinase functions mediate viral cytoplasmic secondary envelopment

Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function enhancing the efficacy of viral tegumentation and release.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65

UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.     

Human cytomegalovirus UL97 D605E polymorphism has a high prevalence in immunocompetent Japanese infants and children

There is no existing data on UL97 mutation in human cytomegalovirus (HCMV) isolates obtained from individuals who have never been exposed to ganciclovir (GCV). UL97 codons 439 to 645 from 61 CMV isolates from 61 immunocompetent Japanese infants and children were sequenced directly. No known GCV resistance mutations were found, meaning that the UL97 mutation had resulted from the use of GCV. On the other hand, a mutation at codon 605 (D to E) was frequently identified (56/61: 91.8%). This could be a genetic marker for HCMV in East Asian counties, because of its low prevalence in the strains of HCMV circulating in Western countries.     

Human Cytomegalovirus Clinical Strain-Specific microRNA miR-UL148D Targets the Human Chemokine RANTES during Infection

The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b'' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.     

Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus

Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.     

The human cytomegalovirus-specific UL1 gene encodes a late-phase glycoprotein incorporated in the virion envelope

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.     

HCMV基因在人和小鼠胚胎成纤维细胞中表达的差异性研究

人巨细胞病毒（human cytomegalovirus, HCMV）种属特异性机制尚不清楚。研究通过检测HCMVADl69体外感染人胚胎成纤维细胞（Human embryo fibroblast, HEF）和小鼠胚胎成纤维细胞（mouse embryo fibroblast, MEF）后病毒基因的表达情况,探讨HCMV种属特异性的可能分子机制。首先用HCMV AD169（MOI=5）分别感染HEF和MEF,相差显微镜逐日观察细胞的形态学变化;RT—PCR检测HCMV即刻早期（IE1、IE2）、早期（uL84）和晚期基因（UL83）的表达情况;Western—blot和免疫荧光检测病毒基因编码蛋白表达的情况。形态学观察发现HEF感染HCMV后逐渐变大变圆并相互融合,第4天可见典型的HCMV特征性病变效应,而MEF则未出现上述的变化;RT-PCR和Western—blot表明HEF组表达即刻早期基因IE1和IE2、早期基因uL84和晚期基因UL83 mRNA以及各基因所编码的蛋白,且相对表达量显著高于模拟感染组（P〈0．01）;而MEF组仅IEl和IE2mRNA和蛋白相对表达量显著低于HEF组（P〈0．05）,而高于模拟感染组（P〈0．01）。免疫荧光检测发现HEF感染72h表达IE和UL83蛋白,而MEF则无明显表达。以上结果表明,HC—MV不能在MEF中复制并产生完整子代病毒颗粒,且病毒基因表达阻止在IE2基因表达之后和UL84基因表达之前,其种属特异性可能与即刻早期蛋白低水平的表达量有关。