A human papilloma virus type 11 transcript encoding an E1--E4 protein

The human papilloma virus (HPV) associated with a genital wart (condyloma acuminatum) was determined to be type 11. The majority of the viral DNA molecules were monomeric circles present in the cells at high copy number, as demonstrated by one- and two-dimensional agarose gell electrophoretic separation followed by Southern blot analysis. A cDNA library in phage lambda gt11 was constructed from poly(A)-selected mRNA recovered from the tissue. Recombinant clones corresponding to the most abundant 1.2-kb viral mRNA species detected by Northern blot hybridization and by electron microscopic analysis of R loops were isolated and their nucleotide sequence was determined. Comparison to the prototype HPV-11 DNA sequence revealed that this message consisted of two exons. The promotor-proximal exon spanned nucleotides 716 through 847 and the distal exon included nucleotides 3325 through 4390 or 4392. The mRNAs were alternatively polyadenylated after either of these latter two sites, in both cases following a G and preceding a U residue. Fourteen or sixteen bases upstream from the poly(A) was the hexanucleotide AGUAAA, which apparently serves as the signal for cleavage and polyadenylation of the nascent message. The splice donor and acceptor sites conformed to the usual /GU. . .AG/pattern. The exons joined open reading frame (ORF) E1, which contributed the initiation codon and four additional triplets, to ORF E4, which specified 85 amino acids to encode a protein of 10,022 Da. The cDNA also contained the ORFs E5a and E5b toward the 3' end. The complete sequence of the cDNA revealed three single-base changes from the prototype HPV-11, two resulting in altered amino acids in E4. Neither affects the coding potential of the overlapping E2 ORF. The function of the E1--E4 protein is unknown.     

Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2

Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.     

Three novel deletions in the alanine:glyoxylate aminotransferase gene of three patients with type 1 hyperoxaluria.

We describe three novel deletions in the human AGT gene in three patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme, alanine glyoxylate aminotransferase (AGT; EC 2.6.1.44). A deletion of 4 nucleotides in the exon 6/intron 6 splice junction (679-IVS6+2delAAgt) is expected to cause missplicing. It would also code for a K227E missense alteration in any mRNA successfully spliced. A 2-bp deletion in exon 11 (1125-1126del CG, cDNA) results in a frameshift. A deletion of at least 5-6 kb, EX1 EX5del, spanned exons 1-5 and contiguous upstream sequence. All three deletions are heterozygous with previously documented missense mutations; the intron 6 deletion with F152I, the exon 11 deletion with G82E, and EX1 EX5del with the common mistargeting mutation, G170R.     

A major transcript of human papillomavirus type 16 in transformed NIH 3T3 cells contains polycistronic mRNA encoding E7, E5, and E1^E4 fusion gene

We have cloned cDNA of the major 1.8 kb mRNA from HPV 16-transformed NIH 3T3 cells (PM3T3). The entire nucleotide sequences of this cDNA were determined and compared with prototype HPV 16 genomic DNA sequences. The 5'-end of the cDNA was flanked by approximately 300 bp of cellular sequences, and the 3'-end of the cDNA sequences contained poly A residues following at nt 4230. HPV 16 sequences began at nt 124, downstream of a major viral p97 promoter, within the E6 open reading frame (ORF). The first splice donor site was at nt 226 and the splice acceptor site was at nt 409, suggesting that the E6 gene is inert. Second splice donor and acceptor sites were located at nt 880 and at nt 3357, respectively. This mRNA was thus shown to consist of three exons, resulting in polycistronic mRNA containing three potentially functional virus early genes--E7, E1--E4, and E5--actively transcribed in the transformant.     

Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.     

Molecular Cloning and Nucleotide Sequence Analysis of a Novel Human Papillomavirus (Type 82) Associated with Vaginal Intraepithelial Neoplasia

The genome of a novel human papillomavirus (HPV-82) was cloned from a vaginal intraepithelial neoplasia grade I. In our series of 291 biopsy specimens, HPV-82 was identified in one case each of cervical intraepithelial neoplasia grade II and grade III by blot hybridization. The histological localization of HPV-82 DNA in the three lesions was confirmed by in situ hybridization. The results indicated that HPV-82 is an etiologic agent for vaginal and cervical intraepithelial neoplasia. By nucleotide sequence similarity of L1 open reading frame (ORF), HPV-82 was closely related to HPV-26, -51, and -69. To know the precise relationship between the HPVs, we determined the complete sequence of HPV-82, as well as that of HPV-69. Sequencing revealed that the four HPVs had no initiation codon in the E5 ORF and had extensive nucleotide sequence similarities in all ORFs. In addition, they exhibited unique frame position patterns for ORFs, different from those of the other genital HPVs.     

Molecular cloning and nucleotide sequence analysis of a novel human papillomavirus (Type 82) associated with vaginal intraepithelial neoplasia

The genome of a novel human papillomavirus (HPV-82) was cloned from a vaginal intraepithelial neoplasia grade I. In our series of 291 biopsy specimens, HPV-82 was identified in one case each of cervical intraepithelial neoplasia grade II and grade III by blot hybridization. The histological localization of HPV-82 DNA in the three lesions was confirmed by in situ hybridization. The results indicated that HPV-82 is an etiologic agent for vaginal and cervical intraepithelial neoplasia. By nucleotide sequence similarity of L1 open reading frame (ORF), HPV-82 was closely related to HPV-26, -51, and -69. To know the precise relationship between the HPVs, we determined the complete sequence of HPV-82, as well as that of HPV-69. Sequencing revealed that the four HPVs had no initiation codon in the E5 ORF and had extensive nucleotide sequence similarities in all ORFs. In addition, they exhibited unique frame position patterns for ORFs, different from those of the other genital HPVs.     

Codon usage bias and A+T content variation in human papillomavirus genomes

We analyzed the codon usage bias of eight open reading frames (ORFs) across up to 79 human papillomavirus (HPV) genotypes from three distinct phylogenetic groups. All eight ORFs across HPV genotypes show a strong codon usage bias, amongst degenerately encoded amino acids, toward 18 codons mainly with T at the 3rd position. For all 18 degenerately encoded amino acids, codon preferences amongst human and animal PV ORFs are significantly different from those averaged across mammalian genes. Across the HPV types, the L2 ORFs show the highest codon usage bias (73.2+/-1.6% and the E4 ORFs the lowest (51.1+/-0.5%), reflecting as similar bias in codon 3rd position A+T content (L2: 76.1+/-4.2%; E4: 58.6+/-4.5%). The E4 ORF, uniquely amongst the HPV ORFs, is G+C rich, while the other ORFs are A+T rich. Codon usage bias correlates positively with A+T content at the codon 3rd position in the E2, E6, L1 and L2 ORFs, but negatively in the E4 ORFs. A general conservation of preferred codon usage across human and non-human PV genotypes whether they originate from a same supergroup or not, together with observed difference between the preferred codon usage for HPV ORFs and for genes of the cells they infect, suggests that specific codon usage bias and A+T content variation may somehow increase the replicational fitness of HPVs in mammalian epithelial cells, and have practical implications for gene therapy of HPV infection.     

Cervical Cancer-causing Human Papillomaviruses have an Alternative Initiation Site for the L1 Protein

All known sequences of the DNA encoding the major cervical cancer-causing human papillomavirus type 16 (HPV16) L1 capsid protein contain initiation codons which would allow translation to begin at either nucleotide 5559 or 5637. However the formation of virus-like particles (VLPs) only occurs efficiently when the initiation codon at nucleotide 5637 is used for in vitro expression studies. This knowledge, in concert with the fact that virions have not been observed in HPV16-infected epithelium, raises the notion that the major L1 translation product in this HPV type may be largely confined to initiation at nucleotide 5559. Sequence analysis of various HPV types associated with particular clinical outcomes has revealed that L1 sequences of the major cervical cancer-associated viruses generally possess the ability to encode a longer translation product whilst the non-cancer-causing viruses do not. Equally intriguing, the upstream initiation codon is always separated by 78 nucleotides from the initiation codon that produces L1 protein which efficiently assembles into VLPs. We speculate that the longer L1 protein could play a role in the development of cervical carcinoma and that HPVs with the potential to cause cervical cancer may be identified by the presence of an in-frame ATG situated 78 nucleotides upstream. An erratum to this article is available at .     

A G-->A transition creates a branch point sequence and activation of a cryptic exon, resulting in the hereditary disorder neurofibromatosis 2

We describe a G-->A transition within intron 5 of the NF2 gene. This mutation creates a consensus splice branch point sequence. To our knowledge this is the first report of a mutation that creates a functional branch point sequence in a human hereditary disorder. The new branch point sequence is located 18 bp upstream of a consensus splice acceptor site. A consensus splice donor site is found 106 bp 3' of the acceptor site. Asa consequence the G-->A transition results in an alternatively spliced mRNA containing an additional exon 5a of 106 bp derived from intron sequences. We cloned the mutant cDNA and show that due to an in-frame stop codon the cDNA codes for a truncated NF2 protein. The mutation was observed in three affected members of an NF2 family. In a tumour of one of the family members both alternatively spliced and wild-type mRNA were found, although the wild-type allele of the gene is absent due to an interstitial deletion on chromosome 22. We also show that immunoprecipitations reveal the presence of full-length wild-type NF2 protein in the tumour lysate. These data support the hypothesis that some degree of normal splicing of the mutant precursor RNA is taking place. It is therefore likely that this residual activity of the mutant allele explains the relatively mild phenotype in the family. These data also indicate that complete inactivation of the gene is not required for tumour formation.      

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.