MATERIALS AND METHODSVirus Isolates. BaMV isolate V (BaMV-V) contains satBaMV (14). BaMV-L is an isolate derived from BaMV-V and free of satBaMV (designated as

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RNAs transcribed from a full-length infectious cDNA clone of the bamboo mosaic potexvirus (strain O) genome, pBaMV-O, were infectious toNicotiana benthamiana plants. Mutant genomes in which the poly(A) tail is absent or replaced by a 3′ tRNA-like structure from turnip yellow mosaic virus RNA failed to amplify detectably in N. benthamiana protoplasts. No amplification was detected in protoplasts inoculated with transcripts containing 4, 7, or 10 adenylate residues at the 3′ end, whereas transcript inocula with 15 adenylate residues resulted in coat protein accumulation to a level 26% of that resulting from inoculation with transcripts with 25 adenylate residues (designated as wild type). Coat protein accumulation levels of 69 and 98% relative to wild type were observed after inoculation of protoplasts with transcripts bearing poly(A) tails 18 and 22 nucleotides long, respectively. The presence of a putative 3′ pseudoknot structure including at least 13 adenylate residues of the 3′-terminal poly(A) tail was supported by enzymatic and chemical structural analysis. The functional relevance of this putative pseudoknot was tested by mutations that affected basepairing within the pseudoknot. These results support the existence of functional 3′ pseudoknot that includes part of the 3′ poly(A) tail.

The papillomavirus replicative helicase E1 and the origin recognition protein E2 are required for efficient viral DNA replication. We fused the green fluorescent protein (GFP) to the human papillomavirus type 11 E1 protein either in a plasmid with the E1 coding region alone (nucleotides [nt] 832 to 2781) (pGFP-11E1) or in a plasmid containing both the E1 and E2 regions (nt 2723 to 3826) and the viral origin of replication (ori) (p11Rc). The former supported transient replication of an ori plasmid, whereas the latter was a self-contained replicon. Unexpectedly, these plasmids produced predominantly a cytoplasmic variant GFP or a GFP-E1^E4 protein, respectively. The majority of the mRNAs had an intragenic or intergenic splice from nt 847 to nt 2622 or from nt 847 to nt 3325, corresponding to the E2 or E1^E4 messages. pGFP-11E1dm and p11Rc-E1dm, mutated at the splice donor site, abolished these splices and increased GFP-E1 protein expression. Three novel, alternatively spliced, putative E2 mRNAs were generated in higher abundance from the mutated replicon than from the wild type. Relative to pGFP-11E1, low levels of pGFP-11E1dm supported more efficient replication, but high levels had a negative effect. In contrast, elevated E2 levels always increased replication. Despite abundant GFP-E1 protein, p11Rc-E1dm replicated less efficiently than the wild type. Collectively, these observations show that the E1/E2 ratio is as important as the E1 and E2 concentrations in determining the replication efficiency. These findings suggest that alternative mRNA splicing could provide a mechanism to regulate E1 and E2 protein expression and DNA replication during different stages of the virus life cycle.Human papillomaviruses (HPVs) comprise a family of small DNA viruses that infect human keratinocytes in stratified epithelia at mucosal or cutaneous sites. Infections are typically subclinical or benign, but certain lesions can progress to cancers at a low frequency (81, 82). The viral genome consists of a double-stranded, circular DNA of approximately 7.9 kb, and it is normally maintained stably as autonomously replicating plasmids present at a low copy number in the basal cells of the epithelium. Vegetative reproduction depends on squamous differentiation, and elevated levels of viral transcription, DNA amplification, and virion morphogenesis occur sequentially in the lower and upper spinous strata of the epithelia. Progeny virus particles are then shed with the terminally differentiated, superficial cells. Because viral DNA replication depends largely on the cellular DNA machinery and entirely on host enzymes to generate deoxyribonucleoside triphosphate substrates, the viruses encode oncoproteins to reestablish an Sphase state in the postmitotic, differentiated cells (11,12,25).The HPV and bovine papillomavirus (BPV) replication origin recognition protein and transcription factor E2 bind as a dimer with high affinity and specificity to multiple copies of a consensus E2 binding site (E2BS), ACCGN 4 CGGT, in the viral origin of rep...

Purified virions of bamboo mosaic potexvirus-V isolate (BaMV-V) were found to contain three major RNA species, the 6n4 kb genomic RNA and two RNAs of 2n0 and 1n0 kb, in addition to associated satellite RNA (0n85 kb). Results of Northern blot hybridization, primer extension analysis and cDNA sequencing showed that the packaged 2n0 and 1n0 kb RNAs of BaMV-V were subgenomic RNAs. In contrast, in the BaMV-O isolate, only genomic RNA was packaged and encapsidated subgenomic RNAs were not detectable. The transcription initiation sites for

A naturally occurring 1.1 kb RNA was isolated from purified virions of bamboo mosaic potexvirus isolate S (BaMV-S). This RNA is a defective RNA (D RNA) derived from a single internal deletion of the BaMV genome. A cDNA clone representing the complete nucleotide sequence of the BaMV-S D RNA was generated and its nucleotide sequence was determined. The BaMV D cDNA is 1015 nts in length [excluding the poly(A) tail] and consists of two regions corresponding to 867 nts of the 5' terminus and 148 nts of the 3' terminus of the BaMV genomic RNA. BaMV D cDNA contains a single open reading frame (ORF) encoding a putative 29.7 kDa protein comprised of a fusion of the first 258 amino acids of BaMV ORF 1 and the last 2 amino acids of coat protein. The coding capacity of D RNA was verified by in vitro translation of native BaMV-S D RNA and of 1.1 kb RNA transcribed in vitro from the full-length D cDNA. BaMV D RNA can be reproducibly generated by serial passages of BaMV-S in Nicotiana benthamiana and is the first D RNA in the potexvirus group shown to be generated de novo. Alignments of sequences surrounding the 5' and 3' junction borders of reported potexvirus D RNAs reveal a 65.2-84.6% sequence identity, suggesting that common mechanisms for viral RNA recombination are involved in the generation of potexvirus D RNAs.

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