The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3'-kinase in NIH 3T3 cells.

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3'-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

Synthesis and analysis of a 640-bp pol region of novel human endogenous retroviral sequences and their evolutionary relationships.

Nucleotide sequence comparisons of the pol gene among 47 retroelements identified two very conserved regions, separated by a span of approximately 640 bp, that have not been previously reported. A set of mixed oligonucleotide primers, 5'-MOP-2 and 3'-MOP-2, homologous to these two conserved pol regions was constructed for use in detection of retroelements. When MOPs-2 were employed in PCR amplification studies, products of about 0.64 kb in size were amplified from human and mouse genomic DNAs and from HIV-1 proviral DNA, but not from negative control plasmid DNAs. The PCR products amplified with MOPs-2 from human LuC-1 teratocarcinoma cell DNA were subcloned and sequenced. Five clones of approximately 0.64 kb in size were identified, and sequence comparisons with all entries in GenBank indicated that these five clones have highest homology, in a range of 64.31 to 98.65%, with the corresponding pol region of HERV-K10 and HM-16 of the human endogenous retrovirus-K (HERV-K) family. Southern hybridizations at high stringency demonstrated that these five clones are present in all human DNAs tested. The evolutionary relationships of these clones with the equivalent pol region of other retroelements were defined by phylogenetic analyses that placed three clones into the HERV-K family and two clones into a new family of human endogenous retroelements. In addition, clone HERV-(K)73 contains the smaller PV1b pol segment that was reported to be selectively expressed in blood leukocytes of patients with polycythemia vera.

Phylogenetic analyses of 55 retroelements on the basis of the nucleotide and product amino acid sequences of the pol gene.

Comparisons of pol gene nucleotide and reverse transcriptase (RT) amino acid sequences of 47 retroviruses, 3 caulimoviruses, and 5 hepadnaviruses showed that approximately one-third of the gene at the 5' end is much more conserved than other pol regions. The most conserved regions on both the nucleotide and amino acid sequences were chosen for construction of phylogenetic trees. The maximum-parsimony and distance-matrix methods were used for analyses of aligned amino acid sequences; these two methods, and the compatibility method, were used to analyze the aligned nucleotide sequences. Essentially identical majority-rule consensus trees were produced by these different methods from both the pol gene nucleotide and RT amino acid sequences, which divided the 55 retroelements into six major groups. The reliability of the phylogenetic trees was probed with the bootstrapping of 100 replicates of the original sequence alignments. The grouping results were shown to be statistically significant by multiple comparisons with the least-significant-difference procedure.

Restricted expression of new HERV-K members in human teratocarcinoma cells.

Northern hybridization with five HERV-K family members, previously cloned from human teratocarcinoma genomic DNA, indicated that two (HERV-(K)27 and -(K)67) of the five clones are expressed in these teratocarcinoma cells. These two clones are closely related (98.49%), however, and Northern blot hybridization lacks the specificity to distinguish between their respective mRNAs. Therefore, PCR analysis with mixed oligonucleotide primers homologous to conserved retroviral pol gene regions was employed to amplify cDNA synthesized from teratocarcinoma cell RNA. This amplification scheme yielded two novel HERV-K family members, HERV-(K)55 and HERV-(K)91. Clone HERV-(K)55 has approximately 98% nucleotide sequence identity to clones HERV-(K)27 and -(K)67. Subsequent RNase protection assays confirmed the expression of HERV-(K)55 and indicated that clones HERV-(K)27 and -(K)67 were not expressed in these cells. One interpretation is that the HERV-(K)27 and -(K)67 probes detected transcripts of clone HERV-(K)55 or other closely-related elements because of their high homologies. In addition, clone HERV-(K)91, which has approximately 81% nucleotide sequence identity to clones HERV-(K)27, -(K)67, and -(K)55, was obtained only from teratocarcinoma 2102E-Pr cells, but the RNase protection assay showed that this clone is also expressed in other human teratocarcinoma cell lines.