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WtSV40 and its variant EL-SV40 (contains two complementing defective genomes) fail to productively infect human embryonic kidney cells or human fibroblasts. However, early SV40 (E-SV40) genomes can propagate in human cells when complemented by a particular late RF virus (L-RFV) genome or the closely related wtBKV genome. The L-RFV genome (L-RFV clone H) contains a deleted early region, a complete set of BKV capsid genes, and a single SV40 regulatory region (acquired by recombination). In contrast, it was not possible to make the reciprocal genome cross in human cells; late SV40 genomes containing a deleted early region do not complement early RFV or early BKV DNAs. The L-RFV clone H genome was also shown to complement wtSV40 in human cells. However, wtSV40 DNA was rapidly lost and replaced by a defective SV40 genome. The SV40 defective (E-SV40 alpha) contained a deletion of the late region, an intact early region, and paired with L-RFV clone H DNA to form a new hybrid virus. In human cells wtSV40 was also complemented by wtBKV DNA, but after two serial passages SV40 DNA disappeared. These findings indicate that SV40 late or capsid gene sequences, but not SV40 early sequences, generate a block to SV40 growth in human cells. When the SV40 late region is replaced by a RFV or a BKV late region, E-SV40 DNA propagates efficiently in human cells and in some cases more rapidly than wtBKV. Northern blot hybridization indicates that SV40 DNA is poorly transcribed in human cells when the SV40 late region is present.

Wild-type (wt) BK virus was introduced into permissive BSC-1 cells along with either early or late defective SV40 genomes. The defectives contained all of the late (L-SV40) or all of the early (E-SV40) coding sequences. Persistently infected (PI) BSC-1 cultures were established and contained wt BKV DNA and E- or L-SV40 DNA in Hirt supernatants. Each of the BKV/SV40 combinations could be serially passed in BSC-1 cells. Also, DNase I digestion of virus stocks from BKV/E-SV40 infections did not eliminate E-SV40. This suggested that (1) E-SV40 genomes could be packaged in BKV capsids and (2) BKV T antigen acted to stimulate the growth of L-SV40 genomes. During continuous culture of PI BSC-1 cells containing BKV and L-SV40, wt BKV genomes were lost and replaced by a BKV defective. The BKV defective (E-BKV) contained a deletion in the late region, an intact early region, and a duplication of the origin. This combination represents a new papovavirus with a bipartite genome in which the early region is derived from BKV and the late region from SV40, and both are present in separate molecules. The BKV and SV40 defectives complement each other for infectivity. Infectious virus is formed with the E-BKV genomes packaged in SV40 capsids. It is hypothesized that this kind of recombination (reassortment) is a way in which papovaviruses may generate variation. The host range for the new BKV/SV40 is narrow. It propagates well in BSC-1 cells, relatively poorly in fetal human brain cells, and not at all in green monkey TC-7 or human embryonic kidney cells. However, it transforms fetal human brain cells at a frequency 25-50 times greater than wt BKV does.

EL SV40 and RFV are variants of SV40 and BKV which contain bipartite or dual genomes. One molecule contains all the early viral sequences (E-SV40, E-RFV) and the other all the late viral sequences (L-SV40, L-RFV). Early and late genomes complement one another during productive infection. Experiments were designed to determine if E-genomes of one virus could complement L-genomes of another virus. If complementation did occur, intermolecular recombination events which lead to a more efficient infection or an altered host range might occur, and the sequences involved could than be identified. Two combinations were generated by direct transfection of BSC-1 green monkey cells. E-RFV and L-SV40 DNA complementation resulted in hybrid virus growth and cell killing. The hybrid demonstrated a narrow host range. Following serial passage, some E-RFV genomes contained SV40 origin region sequences but these recombinants did not overgrow prototype E-RFV genomes, even after many virus passages. In addition, no significant alterations in host range could be detected. Complementation between E-SV40 and L-RFV yielded a virus with a relatively wider host range. Virus growth and cell killing appeared very slowly at first. However, with each passage of E-SV40/L-RFV, cell killing occurred progressively more rapidly, until passage 7 when it became extensive in 7 days rather than 6-8 weeks. Infected cells contained 10-20 times more E-SV40 than L-RFV DNA during the first passage. However, by passage 7, both genomes were equally represented. During serial passage, L-RFV DNA acquired SV40 sequences from around the origin and the terminus of replication, such that recombinant (r) L-RFV genomes contained three SV40 origins [corrected] (including the 72-bp repeat) and 2 termini, and prototype L-RFV DNA was lost. E-SV40/rL-RFV demonstrated an altered host range propagating in some cell lines which did not support E-SV40/L-RFV growth. Both the host range change and the increased growth of rL-RFV genomes were shown to be at least partly caused by the acquisition of the SV40 sequences.

Molecularly cloned viral DNA from late RFV (L-RFV) and early JCV (E-JCV) were transfected into human fetal brain (HFB) cells and complementation was demonstrated. A new infectious virus (E-JCV/L-RFV) was produced. Infection resulted in partial transformation of HFB and human embryonic kidney cells. No transformation was observed with EL-JCV or wtJCV. The transformants contained T-antigen and had a lifespan similar to SV40-transformed human cells but failed to express some phenotypes of transformation. All transformants contained E-JCV viral DNA, usually both integrated and episomal. Although no L-RFV DNA was present in the transformants, L-RFV appears to play a role in the initiation of transformation.