The Study Of The Polymorphism Of Human Cytomegalovirus UL133 Gene In UL/b'region

ObjectiveHuman cytomegalovirus belongs to subfamily Betaherpesvirinae. Con - genital infection of HCMV may cause serious congenital developmental malformation and congenital diseases, such as hepatitis syndrome, congenital megacolon, jau-dice, microcephaly and so on. It can also appear as asymptomatic infection. The complete genome of HCMV consists of 230 ~ 235 Kb double - stranded DNA, containing over 220 open reading frames. Recent research revealed that, the different clinical manifestations of HCMV infection might be correlated with the immune level of the hosts in one hand, on the other hand it might have relationship with the polymorphisms of the genes and their products. In 1996, Cha et al. found that there were 19 ORFs ( UL133 - UL151, located in the UL/b're-gion of HCMV) in the low passaged clinical isolates, while they were absent in the laboratory strain AD169. it was presumed that the genes in the UL/b'region might be lost or rearranged in the reduplicative propagation and passage of the HCMV laboratory strains and led to the obvious decrease of the duplication level, virulence and pathogenesis. It is indicated that the 19 ORFs might play important role in the pathogenesis way of HCMV infection. In this study, we used the nest PCR and DNA sequencing methods, to study the polymorphisms nucleo-tide and amino acid sequences of UL133 gene in HCMV UL/b'region.Materials and Methods1. Patients and specimens22 HCMV clinical low passaged isolates were recovered from the urine or abnormal colon tissue of infants aged less than 14 months with suspected HCMV congenital infection from 1988 to 1993. The clinical manifestations included jaundice (J, n = 12) , microcephaly (M, n=6) and congenital megacolon (C, n =4). QPCR tested HCMV DNA in all isolates positive in 2000. Other virus infections were excluded.Another 11 specimens from HCMV infected congenital megacolon children were also applied in the study.2. Preparation of DNAVirus DNA was exposed from the infected cells by boiling the cell with lysis buffer for 15 minutes.Total DNA of surgical specimens of colon tissue was extracted with xylene, proteinase K, and phenol chloroform protocol, followed by ethanol precipitation. All DNA preparations were diluted in nuclease - free water and kept at 70 °C for use as templates in PCR amplification.3. Selection and synthesis of amplification primersAccording to the UL133 gene nucleotide sequence in certain region of the reference strain Toledo, the nest PCR primers for HCMV UL133 gene were designed using primer designing soft Primer Premier 5.0.4. PCR amplification4. 1 PCR reaction system: The reaction mixture was prepared as follows: 3. 5jlxI sample was added to a 50jxl reaction mix containing 1 x Buffer, 1. 5mM Mgcl2, 0. 2mM dNTP for each, 150 ng forward and reverse primer respectively, and 0. 5U Taq polymerase.4. 2 PCR conditions: nest PCR was performed in this study.4.3 PCR results of HCMV UL133 gene;8^1 of the PCR products were mixed with 2 jxl loading buffer, and visualized on 1.5% agarose gels containing EB with a DNA mass ladder by electrophoresis for 30min under consistent 80V in 1 TBE buffer.5. The purification, recovery and sequencing of the UL133 amplicons The amplicons were purified for sequencing by recovering them from a 1.5% agarose gel using the PCR Fragment Recovery kit (Takara). Purified frag-ments were eluted in 50|jd of ddH20 and sequenced directly with BigDye Terminators Cycle Sequencing Kit. Sequencing was carried out on both DNA strands. Sequencing reactions were performed with a PE Applied Biosystems Geneamp PCRSystem 2400 at 96^1 for 10 sec, 50^for 5 sec, and 60T:for 4 min for a total of 30 cycles. The sequencing results were analyzed on an ABI 3700 automated sequencer.6. Analysis of the sequencesSequence chromatograms were analyzed with Chromas program. All nucleo-tide and amino acid sequence analyses were performed using BioEDIT version 5. 0. 0 and DNAStar version 5.1. Identity scores were obtained by performing pair-wise alignments of sequences with MegAlign. The Clustal W algorithm was used to align multiple sequences of nucleotide and predicted amino acid. Phylogenetic tree was performed using DNAStar. The post - translational modifications of predicted protein were identified with the NCBIPROS database.7. Nucleotide sequences accession numbersThe nucleotide sequences of HCMV UL133 gene were submitted to Gen-Bank using Sequin program.Results1. Results of the amplification and sequencing of HCMV UL133 gene22 HCMV clinical low passaged isolates and 11 specimens from megaclon tissues were all amplified positive for the HCMV UL133 gene by nest PCR. A total number of 33 HCMV UL133 gene sequences were obtained and have been deposited in the GenBank database, with accession number: AY323916 -AY323933, AY997678-997692.2. Analysis of the nucleotide and predicted amino acid sequences of HCMV UL133 geneThe nucleotide and deduced amino acid sequences of UL133 of the 33 clinical strains were compared with 9 available published UL133 sequences in the GenBank, which were formed as exogenous group.2. 1 Polymorphism analysis of the nucleotide and predicted amino acid se-quences of UL133 geneThe identity score among all of the UL133 sequences ranged from 88. 6 -100% at the nucleotide level. The strains obtained in this study shared 88. 6 -100% sequence identity, while the sequences previously published shared 94.2 - 100% sequence identity. Compared to the nucleotide sequence of Toledo strain, the nucleotide changes were dispersed within the whole coding sequence, most of which were substitutions. Approximately 25% of the substitutions were no synonymous.A phylogenetic tree was constructed using the nucleotide sequences of the UL133 gene obtained in this study and those previously published in GenBank. The strains did not cluster according to different clinical symptoms.Among all strains the identity score of the HCMV UL133 gene putative proteins varied from 88. 3 to 100% . The divergences of the 9 previously published strains were less than 7. 4% .2.2The quality and structure analysis of HCMV UL133 gene putative proteinThe HCMV UL133 gene putative product was a protein with a molecular mass of 27. 60KD, and the PI value was 7.73 , which indicated the UL133 putative product might be a protein with alkalescence. The first 30 amino acids in the N terminal were formed as a signal peptide, with a cleavage site at the 30 -Gly and 31 -He.2. 3The analysis of the posttranslational modification motifs of HCMV UL133 gene putative productsHCMV UL133 gene putative products were predicted to contain several posttranslational modification motifs. They were dispersed through th ewhole a-mino acid sequences. With Toledo as an arbitrary reference strain, the posttranslational modification motifs of the putative UL133 protein were conserved except for the region from residue 35 to residue 52.Conclusions1. HCMV UL133 gene exists in the HCMV clinical low passaged isolates.2. Though HCMV UL133 gene presents certain polymorphisms at the nu-cleotide and amino acid level, respectively, they are rather conserved.