1 Department of Microbiology and Immunology, The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA
2 Department of Pediatrics, The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA
3 Department of Obstetrics, Gynecology and Women's Health, The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA
4 Department of Epidemiology and Population Health, The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA
Correspondence
Robert D. Burk
burk{at}aecom.yu.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this work are AF436129 (HPV54A) and AF293961 (HPV82A).
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INTRODUCTION |
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The typical HPV genome contains six open reading frames (ORFs) encoding the structural proteins (L1 and L2), proteins that mediate the viral life cycle (E1 and E2) and proteins that regulate host-cell DNA replication and transformation (E6 and E7) (Knipe et al., 2001). E6 and E7 bind the cell-cycle regulators p53 and pRb, respectively. Among highly cancerous types, these oncogenes degrade p53 and pRb, resulting in host-cell immortalization and proliferation (reviewed by Fehrmann & Laimins, 2003
). Some papillomaviruses (PVs) also encode the E4 and E5 genes, whose roles, although largely unknown, seem to involve functions in the later phases of the viral life cycle (Longworth & Laimins, 2004
; Peh et al., 2004
). The E5 ORF is situated between E2 and L2 in typical PV genomes. In most cases, E5 does not overlap with any of its neighbours; however, the E4 ORF is contained completely within E2.
A great deal of effort has been spent studying the evolutionary implications of overlapping reading frames in viruses (Hein & Stovlbaek, 1995; Krakauer, 2000
; Miyata & Yasunaga, 1978
). A fundamental question is how natural selection operates on two protein sequences encoded by the same stretch of DNA. We address this question in the context of HPV E2 and E4, highlighting rates of synonymous and non-synonymous substitution in overlapping reading frames. To this end, we utilized two recently cloned and sequenced viral isolates, HPV AE2/IS39 and HPV AE9. Although initially thought to be novel types (10 % variance in L1 nucleotide sequences) based on sequence analysis of the MY09/MY11 region of the L1 ORF, upon analysis of their complete genomes, HPV AE2/IS39 and HPV AE9 were found to be subtypes (210 % variance in L1 sequences) of HPV82 and HPV54, respectively. In this report, we examine rates of evolution of each ORF by using dN/dS ratios across these subtype pairs and other closely related mucosal types. Closely related genomes allow examination of evolutionary change prior to saturation of genetic changes. We focus specifically on the modular rates of selection exhibited by E2 and a possible explanation for this modularity, in the overlapping ORF, E4.
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METHODS |
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To clone the potentially novel genomes, PCR primers were designed by alignment of closely related HPV genomes using the sequences of the partial L1 region amplified by MY09/MY11 primers. Additional primers were used to amplify the entire genome in fragments by using overlapping PCR amplification, as described previously (Terai & Burk, 2002). PCR products were separated by agarose-gel electrophoresis, stained with ethidium bromide and visualized under UV illumination. After confirmation of appropriate product sizes, each PCR product was purified (Qiagen gel-extraction kit) and ligated into the pGEM-T Easy vector (Promega) according to the manufacturer's instructions. Initially, SP6 and T7 primers flanking each HPV DNA insert were used to determine the nucleotide sequence. Additional primers were designed by sequence walking. Sequencing was performed in the Albert Einstein Cancer Center DNA-sequencing facility. Overlapping fragments were assembled manually and several additional primers were used to clarify sequence ambiguities. The primer sequences used are available from the authors on request.
Pairwise sequence alignments were performed by using CLUSTAL W (Thompson et al., 1994) with a gap cost of 10·0 and the IUB weight matrix. Multiple sequence alignments, providing prototype and subtype phylogenetic context, were generated in the same way. Calculation of overall, ORF-wide non-synonymous and synonymous changes and rates of non-synonymous and synonymous change were done by using SNAP (Synonymous/Non-synonymous Analysis Program) (Korber, 2002
; Nei & Gojobori, 1986
) and K-Estimator was used to perform sliding-window analysis of dN/dS (Comeron, 1999
).
Phylogenetic trees were constructed in PAUP (version 4.10) (Swofford, 1998) from multiple sequence alignments of the E6, E7, E1, E2, L2 and L1 concatenated ORFs, using both distance (neighbour-joining) and parsimony methods. Alignment gaps were coded as missing before distance and parsimony trees were reconstructed by using equal-weighted characters and 100 bootstrap replicates. To ensure adequate searches in the tree space, 100 random-addition heuristic searches and TBR (tree bisection and reconnection) swapping were employed.
HPVs used in this study and their GenBank accession numbers are as follows: HPV7 (NC_001595), HPV11 (NC_001525), HPV27 (NC_001584), HPV40 (NC_001589), HPV44 (NC_001689), HPV54 (NC_001676), HPV55 (NC_001692), HPV82 (NC_002172), HPV13 (NC_001349), HPV26 (NC_001583), HPV30 (NC_001585), HPV32 (NC_001586), HPV42 (NC_001534), HPV51 (NC_001533), HPV53 (NC_001593), HPV56 (NC_001594), HPV66 (NC_001695), HPV69 (NC_002171), HPV74 (NC_004501), HPV91 (NC_004085), HPV54A (AF436129) and HPV82A (AF293961).
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RESULTS AND DISCUSSION |
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The genomes of HPV54A and HPV82A share a high level of similarity with their prototypes. A comparison of whole genomes reveals 93·5 % nucleic acid similarity between HPV54 and HPV54A, and 89·8 % similarity between HPV82 and HPV82A. The currently accepted criterion for HPV classification relies on comparison of L1 nucleotide ORFs. The L1 ORF in the HPV54 pair diverges by 4·6 %, whilst the HPV82 pair diverges by 7·7 %, placing both isolates within the subtype range.
As expected, subtype and prototype genome lengths are also comparable. HPV82 and HPV82A are 7871 and 7904 bp, respectively, whilst HPV54 and HPV54A are 7759 and 7717 bp in length. Each pair contains the anticipated PV ORFs, including the E6 and E7 proteins, the replication proteins (E1 and E2) and components of the viral capsid (L1 and L2). The only major difference in genomic architecture across the two pairs is HPV54's lack of an E5 homologue. HPV82 contains an unambiguous E5 ORF, but lacks a proximal start codon, whereas the corresponding genomic region in HPV54 is as variable as some sections of its upstream regulatory region. Lack of evidence for E5 in HPV54 reinforces its status as a unique outlier, rooting members of the -PV species clades 1, 8 and 10 (Fig. 1
), all of which contain an intact E5 ORF. The identification of an HPV54 subtype implies that this ancient part of the tree still appears to be evolving. In their greater phylogenetic context, HPV82A and HPV54A sort into species groups 5 and 13, respectively. This evolutionary relationship held in phylogenies constructed from either the concatenated ORFs or L1 nucleotide sequences alone (data not shown) (Fig. 1
).
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ACKNOWLEDGEMENTS |
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Received 5 November 2004;
accepted 8 February 2005.