Analysis of early region 4 of porcine adenovirus type 3

The early region 4 (E4) of porcine adenovirus (PAdV)-3, located at the right-hand end of the genome is transcribed in a leftward direction and has the potential to encode seven (p1-p7) open reading frames (ORFs). To determine the role of each protein in viral replication, we constructed full-length PAdV-3 genomic clones containing deletions of individual E4 ORF or combined deletions of the neighboring ORFs. Transfection of swine testicular (ST) cells with individual E4 mutant plasmid DNAs generated PAdV-3 E4 mutant viruses except with plasmids containing a deletion of ORF p3, ORF p2+ p3 or ORF p3+ p4. Each of the mutants was further analyzed for growth kinetics, and early/late protein synthesis. Mutant viruses carrying deletions in ORF p1, ORF p2 or ORF p4 showed growth characteristics similar to that of wild-type PAdV-3. Early/late protein synthesis was also indistinguishable from that of wild-type PAdV-3. However, mutant viruses carrying deletions in ORF p5, ORF p6 or ORF p7 showed a modest effect in their ability to grow in porcine cells and express early proteins. These results suggest that the E4 ORF p3 (showing low homology with non-essential human adenovirus (HAdV)-9-E4 ORF1 encoded proteins) is essential for the replication of PAdV-3 in vitro. In contrast, the E4 ORF p7 (showing homology to essential HAdV-2 34 kDa protein) is not essential for replication of PAdV-3 in vitro. Moreover, successful deletion of 1.957 kb fragment in E4 region increased the available capacity of replication-competent PAdV-3 (E3 + E4 deleted) to approximately 4.3 kb and that of replication-defective PAdV-3 (E1 + E3 + E4 deleted) to approximately 7 kb. This is extremely useful for the construction of PAdV-3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.     

Genomic and phylogenetic analyses of murine adenovirus 2

Murine adenoviruses (MAdV) are supposedly the oldest members of the genus Mastadenovirus. Currently, there are three distinct MAdV types known with rather different tropism and pathology. Here we report and annotate the DNA sequence of the full genome of MAdV-2. It was found to consist of 35,203 bp thus being considerably larger than the genomes of the other two MAdV types. The increased size of the MAdV-2 genome is generally due to larger genes and ORFs, although some differences in the number of ORFs were observed for the early regions E1, E3 and E4. The homologue of the 19K gene of E1B from MAdV-2 codes for 330 amino acids (aa) and is almost twice as large as from other mastadenoviruses. Accordingly, only the N-terminal half (155aa) has homology to the 19K protein. A homologue of the gene of the 12.5K protein was identified in the E3 region of MAdV-2, but not in MAdV-1 or MAdV-3. The other gene of yet unknown function in the E3 region of MAdV-2 seems to be unique. The E4 region of MAdV-2 contains three ORFs. One has similarity to the 34K gene of other AdVs. Two unique ORFs in the E4 region of MAdV-2 have no homology to any of the five and six ORFs in the E4 region of MAdV-1 or MAdV-3, respectively. Phylogenetic analyses showed that the three murine AdVs have a close common ancestor. They likely formed the first branching of the lineage of mastadenoviruses, and seem to be the most ancient representatives of this genus.     

Detection by antibody probes of human papillomavirus type 6 E5 proteins in respiratory papillomata

We have demonstrated the expression of proteins arising from the E5a and E5b open reading frames (ORFs) of human papillomavirus type 6c (HPV-6c) in respiratory tract papillomata. Recombinant plasmids were constructed to express the ORFs in the bacterial vectors pATH and pRIT2T. Fusion proteins were purified and injected into rabbits to produce polyclonal antibodies. Characterized antibodies generated against these fusion proteins were used in immunoperoxidase assays to identify the presence and distribution of HPV-6 E5 proteins in biopsy specimens of respiratory tract papillomata. The results showed that the E5a and E5b proteins were distributed throughout the thickness of the epithelium in the papillomata but not in the basal layer. The proteins were found in nuclei and in the cytoplasm of koilocytotic cells. Positive reactivity with a similar distribution in the epithelium and subcellular location was obtained in papillomata induced by other HPV-6 subtypes. This cross-reactivity was not unexpected, since nucleotide and amino acid (aa) sequence comparisons between HPV-6c and -6e demonstrated 79% sequence identity with 15 aa substitutions in the 91 aa of E5a. The E5b ORF of HPV-6c has the potential to encode a protein of 74 aa that differed at 28 positions compared with the 72 aa of HPV-6e.     

The nucleotide sequence of adenovirus type 11 early 3 region: comparison of genome type Ad11p and Ad11a.

The early 3 region (E3) of two strains (genome type Ad11p and Ad11a) of human adenovirus serotype 11, causing persistent urinary and acute respiratory illnesses, respectively, has been identified and partially sequenced. The sequenced E3 regions of Ad11p and Ad11a were 1980 and 1966 bp long and encoded three complete ORFs, 18.5, 20.3, 20.6k within the Ad11p genome and 18.5, 20.3, 20.2k within the Ad11a genome. The sequence analysis of the 18.5k gene product demonstrated that a transmembrane domain and a cytoplasmic domain of Ad11p, Ad11a, and Ad35 was identical. Ad11p and Ad35 were homologous in the signal sequence. There was one amino acid mismatch between Ad11p and Ad11a, represented by an alanine instead of a proline. The endoplasmic reticulum lumenal domain, which binds to class I MHC, was relatively conserved between Ad11p and Ad11a with the exception of Glu80 and Glu104 in Ad11p, which were replaced by Gln80 and Lys104 in Ad11a. Within the 20.2k protein of Ad11a, the amino acid sequence Thr-Thr-Ser-His was deleted from a position immediately upstream the transmembrane region of the Ad11p 20.6k protein. The 9.0k E3 open reading frame (ORF) of Ad3 was deleted in the genomes of Ad11p and Ad11a. It is noteworthy that Ad11p and Ad35 which both cause persistent infection of the urinary tract display a remarkable similarity in several ORFs of the E3 region.     

Identification of a Gene Cluster within the Genome of Chilo Iridescent Virus Encoding Enzymes Involved in Viral DNA Replication and Processing

The nucleotide sequence of the genome of Chilo iridescent virus (CIV) between the genome coordinates 0.974 and 0.101 comprising 27,079 bp was determined. Computer-assisted analysis of the DNA sequence of this particular region of the CIV genome revealed the presence of 42 potential open reading frames (ORFs) with coding capacities for polypeptides ranging from 50 to 1,273 amino acid residues. The analysis of the amino acid sequences deduced from the individual ORFs resulted in the identification of 10 potential viral genes that show significant homology to functionally characterized proteins of other species. A cluster of five viral genes that encode enzymes involved in the viral DNA replication was identified including the DNA topoisomerase II (A039L, 1,132 amino acids (aa)), the DNA polymerase (ORF A031L, 1,273 aa), a helicase (ORF A027L, 530 aa), a nucleoside triphosphatase I (ORF A025L, 1,171 aa), and an exonuclease II (ORF A019L, 624 aa), all ORFs possessing the same genomic orientation. The DNA polymerase of CIV showed the highest homology (24.8% identity) to the DNA polymerase of lymphocystis disease virus lymphocystis disease virus 1 (LCDV-1), a member of the family Iridoviridae, indicating the close relatedness of the two viruses. In addition, four putative gene products were found to be significantly homologous to previously identified hypothetical proteins of CIV.     

7型腺病毒疫苗株87mu-97.4mu片段核苷酸序列分析

目的获得７型腺病毒（Ａｄ７）疫苗株８７ｍｕ－９７．４ｍｕ核苷酸序列，分析该区段的基因结构和功能。方法应用Ｓａｎｇｅｒ双脱氧法进行核苷酸序列分析。结果Ａｄ７疫苗株８７ｍｕ－９７．４ｍｕ全长３６９８个核苷酸，推测编码纤维蛋白（３２５个氨基酸）和Ｅ３区１５．４ｋＤ蛋白，Ｅ４区５个蛋白（ＯＲＦ１４．２，ＯＲＦ１５．７，ＯＲＦ８．１，ＯＲＦ４２．８和ＯＲＦ１０．３）。结论这一结果为阐明Ａｄ７的基因结构与功能及利用该病毒做为基因工程疫苗载体打下一定的基础     

Recombination and phylogeographical analysis of Lily symptomless virus

The complete genomic nucleotide sequence of an Indian isolate of Lily symptomless virus (LSV) was determined by sequencing 11 overlapping cDNA fragments of different sizes. The genome consisted of 8,394 nucleotides, excluding the poly (A) tail and contained six open reading frames (ORFs) coding protein for ORF1 220 kDa [1,948 amino acid (aa)], ORF2 25 kDa (228 aa), ORF3 12 kDa (106 aa), ORF4 7 kDa (64 aa), ORF5 32 kDa (291 aa) and ORF6 16 kDa (140 aa) from 5' to 3' end. Sequence was analyzed with other previously characterized full genomes of LSV. Phylogenetic analysis on the basis of RNA-dependent RNA polymerase (RdRp), Triple gene block proteins (TGB's), Coat protein (CP), and ORF6 (16 kDa protein) amino acid sequence revealed that Indian isolate is closely related to The Netherlands Isolate (AJ564638). The overall genome of the present LSV isolate shares 97-98% nucleotide sequence homology with the previously characterized isolates. The phylogenetic analysis, sequence alignment studies, and recombination detection program (RDP3) analysis provided evidence for the occurrence of recombination between the present isolate (AM422452) as major parent and The Netherlands Isolate (AJ564638) and Chinese isolate (AM263208) as minor parents in two different independent recombination events. Based on the recombination analysis, it is suggested that the 3' end of the present isolate is involved in recombination with Chinese isolate (AM263208) and gave rise to the Korean isolate. To the best of our knowledge, this is the first report of complete nucleotide sequence from India and also the first evidence of homologous recombination in LSV.     

Characterization of the early region 3 and fiber genes of Ad7

The nucleotide sequence and the predicted amino acid sequences for open reading frames (ORFs) encoded in the Bam-Hl D fragment of Ad7 (Gomen) DNA show an organization and conservation of potential polypeptides between Ad3 and Ad7. Five ORFs encoded within early region 3 (E3) and shared with the corresponding region of Ad3 can be identified; four of these potential coding regions also share homology to ORFs found in E3 of Ad2 and Ad5. The fiber gene of late region 5 (L5) is also apparent within this region; S1 mapping experiments show that the 5' and 3' boundaries of the main exon in fiber mRNA lie at each end of the proposed fiber ORF. The predicted amino acid sequence for Ad7 fiber shares 60% amino acid homology to Ad3 fiber, but only 20% to Ad2 fiber. Surprisingly, there are three regions of partial amino acid homology near the N- and C-termini of the predicted fiber gene sequences from Ad2, Ad3, Ad5, and Ad7; these conserved regions may be important for interaction with penton base, for proper folding of the shaft of the molecule, or for recognition of the cellular receptor to which adenovirus attaches during infection.     

Functional dissociation of transforming genes of human papillomavirus type 16

Human papillomavirus (HPV) type 16 is thought to be responsible for development of cervical carcinomas, but the mechanism of its carcinogenic action is unknown. To determine which viral genes are involved in cellular transformation, we constructed recombinant murine retrovirus DNAs containing various subgenomic fragments of the HPV 16 early region and examined their transforming activities. The results show that the E6 and E7 ORFs are the transforming genes of HPV 16; the former governs the tumorigenicity in nude mice and the latter influences cell growth properties such as saturation density and colony formation in soft agar. There may also be a tumor-suppressing gene in the E1-E2 ORF region, because the tumorigenic activity of recombinant DNA containing the E1 ORF and the 5' portion of the E2 ORF in addition to the E6 and E7 ORFs was much lower than that of recombinant DNA containing only the E6 and E7 ORFs.     

诺如病毒广州株GZ20133135全基因组序列测定及分析

目的 分析GⅡ-4型诺如病毒广州株GZ20133135全基因组序列,了解其分子结构特点及基因组类型.方法 根据Sydney2012参考株全序列设计引物,用RT-PCR分段扩增诺如病毒全基因组,经分子克隆、测序后进行系统进化分析.结果 GⅡ-4型NoVs广州株GZ20133135病毒基因组全长7566 bp,病毒基因组分三个开放阅读框（ORFs）,ORF1、ORF2及ORF3长度分别为5100 bp、1623 bp和807 bp,ORF1与ORF2之间有19个核苷酸重叠.广州株GZ20133135基因组核苷酸序列与参考株GⅡ-4型Sydney2012变异株同源性最高,全长同源性为99.07％.根据系统进化分析,广州株GZ20133135株属于GⅡ-4 Sydney2012变异株.通过对衣壳区氨基酸比对,Sydney2012变异株位点变化情况为：aa294V或A或P→T,aa296S或T→S,aa297H或Q→R,aa298D或N或T→N,aa368T或N或S或A→E,aa372 N或S→D,aa393 N或D或S,aa394 T或G或S→T,aa395T或A→T,aa407 N或D→S,aa412T或D→N,aa413G或I→T.结论 诺如病毒广州株GZ20133135与参考株Sydney2012NSW0514同源性最高,属于GⅡ-4Sydney2012变异株.全长序列可应用于流行病学诊断、疫苗开发、预测新变异株的出现及诺如病毒流行株进化研究.     

The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex

Nuclear factor E2F is a cellular protein that binds to the adenovirus E2 promoter and E1A enhancer regions and to the cellular c-myc P2 promoter region. The DNA binding activity of E2F, detected in vitro using nuclear extracts prepared from HeLa cells, is increased by adenovirus infection (termed E2F induction). We demonstrate here that a 19.5-kD protein, encoded by adenovirus early region 4 (E4) open reading frame (ORF) 6/7, is primarily responsible for the induction of E2F DNA binding activity to the E2 promoter region. Viral mutants that contain frame-shift mutations in E4 ORF 6/7 failed to induce E2F binding activity; a virus that carries an E4 ORF 6/7 cDNA in place of the E4-coding sequences induced E2F efficiently. Using gel mobility shift assays, we demonstrate that the E4 ORF 6/7 product induces the binding of E2F to the E2 promoter via a direct complex. The addition of a peptide-specific antiserum, directed against the E4 ORF 6/7 protein, to an in vitro E2F-binding reaction resulted in the formation of a DNA-protein complex with reduced gel mobility compared to the normal, adenovirus-induced E2F-E2 promoter complex. The formation of the E2F-E2 promoter-antibody complex was blocked by the addition of the cognate peptide used to generate the antiserum but not by a nonspecific peptide. Nuclear extracts prepared from adenovirus-infected HeLa cells were cleared of E2F binding activity using the ORF 6/7 peptide-specific serum, but not the preimmune serum, suggesting that E2F and the E4 ORF 6/7 product form a protein-protein complex in solution. The adenovirus E1A proteins are not absolutely required for the induction of E2F binding activity because the infection of HeLa cells with an E1A mutant, dl312, at high multiplicity resulted in E2F induction. Under these conditions of infection, the E4 ORF 6/7 product was synthesized. E2F binding activity was induced, but inefficiently, in cells infected with E4 ORF 6/7 mutants, indicating that an additional pathway may lead to E2F induction.