Taming of the shrewd: novel eukaryotic genes from RNA viruses

Genomes of several yeast species contain integrated DNA copies of complete genomes or individual genes of non-retroviral double-strand RNA viruses as reported in a recent BMC Biology article by Taylor and Bruenn. The integrated virus-specific sequences are at least partially expressed and seem to evolve under pressure of purifying selection, indicating that these are functional genes. Together with similar reports on integrated copies of some animal RNA viruses, these results suggest that integration of DNA copies of non-reverse-transcribing RNA viruses might be much more common than previously thought. The integrated copies could contribute to acquired immunity to the respective viruses.     

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.     

Expression levels of the human DNA repair protein metnase influence lentiviral genomic integration

We recently identified a Transposase domain protein called Metnase, which assists in repairing DNA double-strand breaks (DSB) via non-homologous end-joining (NHEJ), and is important for foreign DNA integration into a host cell genome. Since integration is essential for productive lentiviral infection we examined whether Metnase expression levels could have an influence on lentiviral genomic integration. Using cells stably transduced to either over- or under-express Metnase we determined that the expression level of Metnase did indeed correlate with live lentiviral integration. Changes in Metnase levels were accompanied by changes in the number of copies of integrated lentiviral cDNA. While Metnase levels affected lentiviral integration, it had no effect on the amount of either total cellular viral RNA, cDNA or 2-LTR circles. Therefore, Metnase enhances the integration of lentivirus DNA into the host cell genome.     

Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors.

The keratinocyte line SK-v harbors only integrated human papillomavirus type 16 (HPV 16) DNA sequences, although it originated from vulvar Bowenoid papules predominantly containing multiple copies of free HPV 16 genomes. We have cloned a fragment of cell DNA that contains the integrated HPV 16 DNA sequences and have shown that integration interrupts the HPV 16 genome in open reading frames E2 and L2 and creates a deletion of 813 base pairs. This allows the expression of open reading frames E6 and E7, as actually substantiated by Northern (RNA) blot analysis of SK-v RNAs with subgenomic HPV 16 RNA probes. Using a unique flanking cellular DNA sequence as the probe, we have shown that the integration of HPV 16 sequences had already occurred in the premalignant lesions from which the SK-v cell line was derived.     

Relationships between the presence of anti-Tat antibody, DNA and RNA viral load

The possible relationships between the intensity of humoral response to full length Tat protein, the amount of proviral DNA reservoir in peripheral blood mononuclear cells and RNA viral load were analyzed in plasma samples obtained from a group of HIV-1 seropositive subjects, who never received any antiretroviral therapy. All HIV-1 patients showed detectable levels of serum IgG to full-length Tat by immunoenzymatic assay. We found a higher percentage of HIV-1 seropositive subjects with low levels of antibody in the presence of barely detectable proviral DNA copies (< or =10 copies/1.5x10(5) PBMCs) and a high anti-Tat antibody response accompanied by variable (from >10(1) to > or =10(3) copies/1.5x10(5) PBMCs) levels of DNA load (p=0.011). Moreover, an inverse relationship between anti-Tat antibody titers and HIV-1 RNA viral load was demonstrated HIV-1 seropositive patients. In HIV-1-infected patients, a strong humoral immune response against HIV-1 transactivating Tat protein, able to down-modulate viral replication in peripheral blood, does not seem to inhibit the number of proviral DNA molecules in peripheral blood mononuclear cells. Even though our data strongly confirm the "positive" role of anti-Tat antibody on viral replication, the persistence of significant amount of DNA viral load in peripheral blood mononuclear cells, despite high level of anti Tat antibody, suggests a more cautious approach to HIV-1 Tat-containing vaccines, able to stimulate an immune specific response to transactivating Tat protein sufficient in inhibiting circulating virus, but not completely efficient in decreasing proviral DNA integration.     

Recognition of duplex DNA by RNA polynucleotides.

We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids. Homopyrimidine RNA oligonucleotides bind to duplex DNA at homopurine/homopyrimidine sequences under slightly acidic conditions. Recognition is sequence-specific, involving rU.dA.dT and rC+.dG.dC base triplets. Affinities were determined for folded polymeric RNAs (ca. 100-200 nt) containing 0, 1 or 3 copies of a 21 nt RNA sequence that binds duplex DNA by triple helix formation. When this recognition sequence was inserted into the larger folded RNAs, micromolar concentrations of the resulting RNA ligands bound a duplex DNA target at pH 5. However, these binding affinities were at least 20-fold lower than the affinity of an RNA oligonucleotide containing only the recognition sequence. Enzymatic probing of folded RNAs suggests that reduced affinity arises from unfavorable electrostatic, structural and topological considerations. The affinity of a polymeric RNA with three copies of the recognition sequence was greater than that of a polymeric RNA with a single copy of the sequence. This affinity difference ranged from 2.6- to 13-fold, depending on pH. Binding of duplex DNA by polymeric RNA might be improved by optimizing the RNA structure to efficiently present the recognition sequence.     

Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells.

Integration of human papillomavirus type 16 (HPV-16) DNA into a host chromosome has been hypothesized to result in altered expression of two viral transforming genes, E6 and E7, in cervical cancers. In order to investigate the role that changes in viral genomic state and gene expression play in cervical carcinogenesis, we have derived clonal populations of human cervical epithelial cells which harbor multiple copies of either extrachromosomal or integrated viral DNA. The clonal populations harboring extrachromosomal HPV-16 DNA stably maintained approximately 1,000 viral copies for at least 15 passages (approximately 100 cell doublings), which contrasted with the unstable HPV-16 replicons in the parental counterpart. In the clonal populations harboring integrated viral DNA, 3 to 60 copies of HPV-16 DNA were found integrated in either of two forms: type 1, in which all the copies of HPV-16 DNA were disrupted in the E2 open reading frame upon integration, and type 2, in which intact viral copies were flanked by disrupted viral copies and cellular sequences. Despite the lower HPV-16 DNA copy number, the clonal populations with integrated viral DNA had levels of E7 protein that were in most cases higher than those found in the clonal populations harboring extrachromosomal viral DNA. Irrespective of viral genomic state, the clonal populations were capable of undergoing terminal differentiation and unable to form colonies in soft agar, which is indicative of the nontumorigenic nature of these cells. Importantly, a cell population with integrated viral DNA was found to outgrow another with extrachromosomal DNA when these cells were cocultured over a period of time. Thus, integration of human papillomaviral DNA correlates with increased viral gene expression and cellular growth advantage. These observations are consistent with the hypothesis that integration provides a selective advantage to cervical epithelial precursors of cervical carcinoma.     

Terminally redundant sequences in cellular intracisternal A-particle genes.

The sequences coding for intracisternal A-particle RNA form a family of related but not identical genetic elements which are present in 650 to 1,000 copies within the mouse genome. We showed that different intracisternal A-particle genes had a terminally redundant sequence of about 400 base pairs, one-half of which arose from the 3' end of the intracisternal A-particle RNA. A second portion of the redundant region did not contain 3-related sequences and was probably derived from the 5' end of intracisternal A-particle RNA. Thus, there were endogenous intracisternal A-particle genes in the cellular DNA-3'-5'--3'-5'-cellular DNA configuration identified for type B and C retroviruses. This indicated that the initial integration of intracisternal A-particle genes into the Mus musculus genome occurred by the same mechanism as the integration of other retroviruses. Two types of heterogeneity were identified among the 5' sequences of the two genes.     

Introduction of stable high-copy-number DNA into Chinese hamster ovary cells by electroporation

A new gene transfer protocol has been developed that introduces up to 800 copies of an expression vector into Chinese hamster ovary cells in a single step by electroporation. The DNA typically integrates in tandem repeats so that the restriction endonuclease site used to linearize the input DNA remains intact. This is likely due to ligation of vector DNA via cohesive ends prior to integration. This high-copy-number procedure is far more rapid than the conventional stepwise gene amplification method used to generate stable eukaryotic protein production cell lines. By employing the expression vector pJODtPA, in which the selectable marker dihydrofolate reductase (DHFR) and the human tissue plasminogen activator (tPA) casettes are separated by a spacer and an RNA polymerase II terminator, cell lines secreting as much as 24 pg/cell.day tPA were isolated following electroporation and a single methotrexate selection. Gene copies and expression levels are stable over long periods of growth. A single round of gene amplification was performed following the high-copy-number procedure to yield a clone having a tPA production level of 45 pg/cell.day.     

Nonrandom integration of human U4 RNA pseudogenes.

Four loci for human U4 RNA have been characterized by DNA sequence analysis. The results show that all four loci represent pseudogenes, which are flanked by direct repeats. Three of the pseudogenes, designated U4/5, U4/6, and U4/8, have very similar structures; they are all truncated and contain the first 67 to 68 nucleotides of the U4 RNA sequence. Their properties suggest that they were created by integration of truncated cDNA copies of the U4 RNA into new chromosomal sites. An interesting observation was that their flanking regions exhibit sequence homology. A purine-rich 5'-flanking sequence 12 to 13 nucleotides long is almost perfectly conserved in all three loci. Boxes of homology were also found on the 3' side when the U4/6 and U4/8 loci were compared. The U4/4 locus has a slightly different structure; the pseudogene matches the first 79 nucleotides of U4 RNA, but contains a greater number of mutations than the other pseudogenes. Taken together, the results suggest that a frequently occurring type of pseudogene for human U4 was created by a RNA-mediated mechanism and that the integration sites have features in common.     

High levels of viral replication during primary simian immunodeficiency virus SIVagm infection are rapidly and strongly controlled in African green monkeys

In contrast to pathogenic human immunodeficiency virus and simian immunodeficiency virus (SIV) infections, chronic SIVagm infections in African green monkeys (AGMs) are characterized by persistently low peripheral and tissue viral loads that correlate with the lack of disease observed in these animals. We report here data on the dynamics of acute SIVagm infection in AGMs that exhibit remarkable similarities with viral replication patterns observed in peripheral blood during the first 2 weeks of pathogenic SIVmac infections. Plasma viremia was evident at day 3 postinfection (p.i.) in AGMs, and rapid viral replication led by days 7 to 10 to peak viremias characterized by high levels of antigenemia (1.2 to 5 ng of p27/ml of plasma), peripheral DNA viral load (10(4) to 10(5) DNA copies/10(6) peripheral blood mononuclear cells [PBMC]), and plasma RNA viral load (2 x 10(6) to 2 x 10(8) RNA copies/ml). The lymph node (LN) RNA and DNA viral load patterns were similar to those in blood, with peaks observed between day 7 and day 14. These values in LNs (ranging from 3 x 10(5) to 3 x 10(6) RNA copies/10(6) LN cell [LNC] and 10(3) to 10(4) DNA copies/10(6) LNC) were at no time point higher than those observed in the blood. Both in LNs and in blood, rapid and significant decreases were observed in all infected animals after this peak of viral replication. Within 3 to 4 weeks p. i., antigenemia was no longer detectable and peripheral viral loads decreased to values similar to those characteristic of the chronic phase of infection (10(2) to 10(3) DNA copies/10(6) PBMC and 2 x 10(3) to 2 x 10(5) RNA copies/ml of plasma). In LNs, viral loads declined to 5 x 10(1) to 10(3) DNA copies and 10(4) to 3 x 10(5) RNA copies per 10(6) LNC at day 28 p.i. and continued to decrease until day 84 p.i. (<10 to 3 x 10(4) RNA copies/10(6) LNC). Despite extensive viremia during primary infection, neither follicular hyperplasia nor CD8(+) cell infiltration into LN germinal centers was detected. Altogether, these results indicate that the nonpathogenic outcome of SIVagm infection in its natural host is associated with a rapidly induced control of viral replication in response to SIVagm infection, rather than with a poorly replicating virus or a constitutive host genetic resistance to virus replication.