Sequence Variabilities Of Human Cytomegalovirus UL139,UL140 And UL138 Genes

Human Cytomegalovirus(HCMV) is a ubiquitous betaherpesvirus with a wide spectrum of infectivity, as demonstrated by several cell types that can be infected in vitro and by multiple organs observed during infection in vivo.HCMV infection can cause serious diseases, particularly in immunocompromised patients and newborns. However, the mechanisms of viral pathogenesis are not known. Differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent HCMV infections are thought to be related to genomic variability among strains.HCMV is a genetically complicated virus, its genome consists of 230-235 kb double-strands DNA and more than 200 predicted open reading frames (ORFs). A new region of HCMV DNA containing at least 19 ORFs( UL133-UL151) was found in the Toledo strain(EMBL,AY446871) and in several other low passage clinical isolates recently, but it was deleted in laborotary strain AD169(EMBL,X17403). The fact that AD169 shows attenuated virulence and different tropisms in endothelial cell from low passage isolates,suggests that predicted products of these new ORFs possibly determine the outcome of HCMV infection in vivo.The possibility that UL133-151 ORFs in the UL/b′region may provide genetic markers for HCMV pathogenesis prompted to investigate these genes in HCMV clinical isolates. UL141 might downregulate natural killer cell-activating ligand CD155, UL144 encodes a structural homologue of the herpesvirus entry mediator and both UL146 and UL147 encode viral CXC(α) chemokines. At present,the variabilities and functions of these genes are being investigated.However, the definitive associations between these genes and HCMV disease have not been established,which leaves open the possibility that other variable HCMV-encoded products in the region may play a role in viral pathogenesis.The genomic variabilities of HCMV UL139,UL140 and UL138 ORF and the relationship between the variabilities of these genes and different pathogenesis of congenital HCMV infection were detailedly investigated in this report.Materials and Methods1.Patients and SamplesThe study population of HCMV UL139 consisted of twenty-six infants with HCMV infection who were identified in the 2nd affiliated hospital of China Medical University. These infants aged less than 14 months with suspected congenital infection had different clinical manifestations: jaundice (J,n=15), Hirschsprung's disease (C,n=4), microcephaly (M,n=4)and asymptomatic infection (A,n=3). Nineteen low passage clinical isolates and seven urine samples from the infants were used to carry out PCR amplification. Isolates, recovered from abnormal colon tissue or urine, were passaged less than 10 times in human embryonic lung fibroblasts (HELF).The study population of UL140 and UL138 consisted of thirty HCMV infected infants who exhibited different clinical manifestations: jaundice (n=18), Hirschsprung's disease (n=7),microcephaly (n=5). All sample were clinical isolates.2.PCR AmplificationAccording to the corresponding Toledo sequence, primers were designed using Primer premier 5.0 software, see Table 1. The PCR reaction mixture contained 1×buffer, 1.5mM MgCl₂, 0.2mM dNTPs, forward and reverse primers 150ng respectively , 0.5U of Taq polymerase,template 3.5μl in a final volume of 50μl.The conditions for amplification were 95℃for 4 min followed by 30 cycles of 95℃for 45 sec, 54℃for 1 min, 72℃for 1 min, and a final extension at 72℃for 10 min.3.Purification and SequencingThe PCR products of the appropriate size were purified for sequencing using the PCR Fragment Recovery Kit. Sequencing was carded out on both DNA strands. The sequences were analyzed on an ABI 3700 automated sequencer.4.Sequence AnalysisAll nucleotide and amino acid sequences were analyzed using the BioEdit 5.0,DNAClub,GeneDoc,DNAStar and Sequin software.RESULTS1.Presence of the UL139, UL140 and UL138 ORFAll samples gave positive amplification of HCMV UL139,UL140 and UL138. PCR products selected were sequenced successfully. The sequences have been assigned Genbank accession No. AY218873-AY218887, AY601874-AY601877, AY8052552, AY805259, AY805260, AY805292, AY805294, AY905263, AY905264, AY218860-AY218872, AY255772, AY255773, AY601873, AY941163-AY941165 and AY218849-AY218859, AY255774-AY255777.2. Analysis of the UL139,UL140 and UL138 DNA and Amino Acid Sequence(1)Analysis of the UL139 DNA and Amino Acid SequenceThe UL139 gene sequences from HCMV infected infants ranged in length from 408 to 444 bp. Using the Toledo strain as the arbitrary reference sequence, the variants were clustered clearly into three major groups G1 (Gla,G1b,G1c), G2 (G2a,G2b) and G3. The strains in Gla showed a high level of identity to the Toledo strain. However, the strains in the other subgroups contained 12-36 nucleotide insertions and 24-49 substitutions, of which 47.8-60.7% were non-synonymous with respect to the Toledo strain.The non-synonymous nucleotide substitutions led to a wide spectrum of amino acid changes. Phylogenetic analysis of the UL139 putative proteins was mainly consistent with the results described for DNA. Although a relatively large number of nucleotides were inserted in the 5' half of the UL139 ORF in some strains, no frame-shift mutation occurred due to 3 or 3N nucleotide insertions. The C-terminal region and the eight cysteines of all UL139 putative protein appeared relatively conservative.(2)Analysis of the UL140 DNA and Amino Acid SequenceThe UL140 ORF in clinical isolates was identical in size to that of Toledo, composed of 576 nucleotides. The identities in nucleotide were 96.8-100.0%. The UL 140 ORF was predicted to be a possible glycoprotein of 191 amino acids. Alignment of amino acid sequences showed a high level of identity from 95.4% to 100.0%.(3)Analysis of the UL138 DNA and Amino Acid SequenceThe UL138 ORF in clinical isolates was identical in size to that of Toledo too, composed of 510 nucleotides and predicted to be a protein of 169 amino acids. The identities in nucleotide and amino acid sequences were 97.6%-100.0% and 98.2%- 100.0% respectively.3.Structure Analysis of the Predicted UL139, UL140 and UL138Proteins(1)Structure Analysis of the Predicted UL 139 ProteinThe UL139 putative protein was predicted to be a glycoprotein(gpUL139). All gpUL139 shared sequence homology with human CD24 mature peptide in a restricted region. The gpUL139 contained 4 or 5 N-glycosylation sites(ASN), 3 or 4 Casein kinaseâ…¡phosphorylation sites(CKP), 2-4 N-myristoylation sites(MYR), depending on the sequence groups which clinical strains belonged to. In addition, the sequences in the strains of Glb contained a specific attachment site of prokaryotic membrane lipoprotein lipid.(2)Structure Analysis of the Predicted UL 140 ProteinThe post-translational modification motifs of UL 140 putative proteins in clinical isolates were highly conserved. 2J, 39J, 27C and 29C added a CKP site in the same position. However, these isolates were derived from the infants with different clinical signs which were jaundice and Hirschsprung's disease respectively.(3)Structure Analysis of the Predicted UL 138 ProteinThe post-translational modification motifs of UL138 putative proteins in clinical isolates were highly conserved.4.Relationship Between UL139,UL140,UL138 and Signs of Congenital InfectionsThe strains from infants with asymptomatic HCMV infection, as those from patients who suffered from HCMV disease,existed in three UL139 genotypes. The distribution of the strains from infants with different clinical manifestations in the UL139, UL140 and UL138 sequence groups was not apparently different.Conclusions1. HCMV UL139, UL140 and UL138 genes were extensively present in HCMV clinical strains.2. HCMV UL139 gene is one of the most variable region in HCMV. A large number of nucleotide insertions and non-synonymous substitutions occurred in the UL 139 ORF. The clustered polymorphisms in the UL 139 sequence imply that selective pressures might favor retention of each subgroup.3. The finding that the putative gpUL139 shared sequence homology with human CD24 suggested that the UL139 putative protein might be involved in the immune response during HCMV infection.4. HCMV UL 138 and UL 140 genes were highly conserved.