In this study we made use of powerful selection schemes to identify cis
elements in the human papillomavirus type 16 genome that support its stable plasmid replication in yeast. A single element within the HPV16 genome, termed the rep
element, that could substitute for a yeast ARS was located to a 1,000-bp region overlapping the 3′ end of the L1 ORF and the 5′ portion of the LCR. This rep
element is positioned close to but separable from the E1-dependent replication origin, E1 ori
, which is positioned at the very 3′ end of the LCR. Consistent with this fact, the rep
element substituted for a yeast ARS in the absence of any HPV genes, including E1, indicating that, like the full-length genome (2
), this element supports plasmid replication in yeast in the absence of the viral E1 DNA helicase.
In a separate study, in which we investigated the capacity of HPV16 DNA to replicate in highly transformed human cell lines independently of E1, a subgenomic DNA fragment that closely overlaps this region of the HPV16 genome replicates in short-term replication assays (K. Kim and P. F. Lambert, unpublished observations). This raises the possibility that sequences within this region of the viral genome can function to support replication in many types of eukaryotic cells. Further studies are necessary to learn whether the same, different, or partially overlapping sets of DNA sequences within this region contribute to replication in both yeast and mammalian cells.
Three separable regions within the HPV16 genome were found to substitute for a yeast CEN element. Two of these elements, mtc2 and mtc3, were identified in multiple independent clones that were rescued from yeast following outgrowth, indicating that they are efficient in supporting stable plasmid inheritance; whereas mtc1 was represented only once among the rescued clones. As predicted, each of the mtc elements restored plasmid stability to a plasmid lacking a centromere (Fig. ). mtc3 and less so, mtc2, caused the ARS+CEN− shuttle vector to replicate at a low copy number in yeast, similar to that seen with yeast plasmids containing a CEN element, as well as with the intact HPV16 genome in yeast. It is not yet clear what sequence elements in mtc2 and mtc3 might confer this property or whether it has relevance to low-copy replication observed in human cells.
elements can substitute for ARS and CEN elements, we analyzed the HPV16 DNA sequence for sequence motifs that are thought to contribute to the function of ARS and CEN elements. Eight imperfect matches to the ARS consensus sequence, [A/T]TTTAT[A/G]TTT[A/T], were identified; each was mispaired by one nucleotide from the consensus (Fig. ). Only the ARS-like sequence within the E1 ORF (nucleotides 1640 to 1650) was predicted to have any activity, that being extremely weak compared to ARS consensus variants (32
) (Fig. ). The Oriscan results concurred that none of the identified ARS-like elements could be predicted to have significant activity. Further confirmation was that the functional ARS screens (Fig. and ) only recovered the region spanning the L1 ORF (nucleotides 6732 to 7793) and not the weak ARS predicted in the E1 ORF (nucleotides 1639 to 1655). Thus, sequence similarity between rep
and ARS elements is insufficient to predict the function of rep
In yeast, not only ARS element clusters but also DNA unwinding elements and AT-rich sequences influence the efficiency of replication (19
). Therefore, a reasonable interpretation is that the region of the viral genome that contains the rep
element provides replicative function owing to the presence of additional undefined sequence elements. Further dissection of the rep
element will be required to identify those relevant DNA sequences and assess of their potential role in the viral life cycle. One possibility is that the replicative function residing within the L1 ORF (nucleotides 6732 to 7793) provides low-copy replication and functions in an adjunct fashion to the E1-dependent origin, which resides at the 3′ end of the LCR. It has been argued that papillomaviruses can exist in a latent infective state. Under such conditions, one might predict that the E1-independent origin could provide the virus with an ability to persist as a nuclear plasmid in the absence of viral gene expression. Testing for such a role would require a laboratory model for latency, which today does not exist.
The positions of the mtc elements in the HPV16 genome are not well explained by the locations of sequence homology to CEN elements CDE I and CDE III (Fig. ). Only the region of the HPV16 genome harboring mtc1 contained two perfect matches, both to CDE I, in addition to one imperfect match, also to CDE I (Fig. ). Yet we infer that this is the weakest of the mtc elements because it was identified in only one of the rescued clones following selection. mtc2 and mtc3, the elements more frequently identified in the selection scheme, have no consensus CDEs, though the region of the viral genome that harbors mtc3 does contain one single mismatched CDE III element (Fig. ). These results suggest that mtc elements may be functioning to support plasmid maintenance by a mechanism that is dissimilar to centromere function. Further dissection of the sequences that confer mtc function and analysis of their role in the viral life cycle is currently under way. It remains unclear whether these mtc elements function in mammalian cells and, if so, act independently of the E2-mediated plasmid maintenance activity or act to augment it.