Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus

Abstract

The sequence data (H. Okamoto et al., Hepatol. Res. 10:1-16, 1998) of a newly discovered single-stranded DNA virus, TT virus (TTV), showed that it did not have the terminal structure typical of a parvovirus. Elucidation of the complete genome structure was necessary to understand the nature of TTV. We obtained a 1.0-kb amplified product from serum samples of four TTV carriers by an inverted, nested long PCR targeted for nucleotides (nt) 3025 to 3739 and 1 to 216 of TTV. The sequence of a clone obtained from serum sample TA278 was compared with those registered in GenBank. The complete circular TTV genome contained a novel sequence of 113 nt (nt 3740 to 3852 [=0]) in between the known 3'- and 5'-end arms, forming a 117-nt GC-rich stretch (GC content, 90.6% at nt 3736 to 3852). We found a 36-nt stretch (nt 3816 to 3851) with an 80.6% similarity to chicken anemia virus (CAV) (nt 2237 to 2272 of M55918), a vertebrate circovirus. A putative SP-1 site was located at nt 3834 to 3839, followed by a TATA box at nt 85 to 90, the first initiation codon of a putative VP2 at nt 107 to 109, the termination codon of a putative VP1 at nt 2899 to 2901, and a poly(A) signal at nt 3073 to 3078. The arrangement was similar to that of CAV. Furthermore, several AP-2 and ATF/CREB binding sites and an NF-kappaB site were arranged around the GC-rich region in both TTV and CAV. The data suggested that TTV is circular and similar to CAV in its genomic organization, implying that TTV is the first human circovirus.

FIG. 1

Seminested short PCR and inverted, nested long PCR for TTV. (A) Schematic diagram of the TTV genome and target regions for the seminested and inverted, nested long PCRs. The dotted region in the genome indicates the region identified in this study. Arrows indicate locations and directions of primers. (B) Seminested short PCR. The products were electrophoresed in a 3% agarose gel and stained with ethidium bromide. The expected sizes of the products of the first and second PCRs were 286 and 271 nt, respectively. (C) Inverted, nested long PCR. The products were electrophoresed in a 1% agarose gel and stained with ethidium bromide. The product sizes of the first and second PCRs were 1.2 and 1.0 kb, respectively. (D) Southern blotting of the gel in panel C probed by the insert of pBS317pma (nt 3706 to 3852 and 1 to 22). Note that the autoradiogram for the first PCR is overexposed. Lanes: M, φX174 HaeIII digest for panel B and λ phage HindIII digest for panel C; 1 and 7, template-free control; 2 and 8, TP100 negative serum; 3 to 6 and 9 to 12, TA278, TP88, TP97, and TP110 sera, respectively; 13 and 14, a cloned TTV DNA at six copies per reaction.

FIG. 2

Sequence strategy. The insert of pTZ27852, 1.0 kb in size, was sequenced from both directions with two series of deletion mutants: the BS series with 5′-end deletions and the NS series with 3′-end deletions. Shown are the area sequenced (not the size of insert) and the sequence direction of representative clones. Open box on the scale, the novel GC-rich region.

FIG. 3

Nucleotide sequences of the pTZ27852 insert. The 1,000 nt between the inner primer pairs were compared with those of the previously reported TTV, both from sample TA278 (GenBank no. AB008394). A portion of the CAV genome (M55918) which had an 80.6% similarity to the TTV genome is also included. Dashes, nucleotide matches with AB008394; asterisks, nucleotide matches with M55918; underline, ATF/CREB site; thick underline, AP-2 site; solid box, SP-1 site; dashed box, NF-κB site.

FIG. 4

ORF analysis of pTZ27852. The upper panel is for the positive strand, and the lower panel is for the negative strand. The arrows indicate the ORFs found (>100 nt) and their directions. Short separator, initiation codon; long separator, termination codon; box in the scale, the novel GC-rich region.

FIG. 5

Comparative scheme of three major ORFs of TTV and CAV. The ORFs of the TTV genome, VP1* through VP3*, are putative. Closed box, the novel GC-rich region; arrow, SP-1 site; open triangle, poly(A) site; closed triangle, TATA box.