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DNA editing using CRISPR/Cas has emerged as a potential treatment for diseases caused by pathogenic human DNA viruses. One potential target is HIV-1, which replicates via a chromosomally integrated DNA provirus. While CRISPR/Cas can protect T cells from de novo HIV-1 infection, HIV-1 frequently becomes resistant due to mutations in the chosen single guide RNA (sgRNA) target site. To address this problem, we asked whether an sgRNA targeted to a conserved, functionally critical HIV-1 sequence might prevent the selection of escape mutants. We report that two sgRNAs specific for the HIV-1 transactivation response (TAR) element produce opposite results: the TAR2 sgRNA rapidly selects for mutants that retain TAR function, but are no longer inhibited by Cas9, while the TAR1 sgRNA fails to select any replication competent TAR mutants, most probably because it is targeted to a region of TAR that is disrupted by even minor mutations. [ABSTRACT FROM AUTHOR]

Abstract: Few cases of acute adult T-cell leukemia/lymphoma (ATL) have been diagnosed in young patients. This report is the first to describe a young girl with infective dermatitis associated with HTLV-1 that progressed to acute ATL with Southern blot hybridization and gamma-TCR-rearrangement revealing a monoclonal pattern with two copies of the provirus. [Copyright &y& Elsevier]

Abstract: The colony of wild mice, named Jyg, has been shown to express an exogenous mouse mammary tumor virus (Jyg-MMTV). This virus induces mammary tumors in its natural host at a high incidence (≈80%) resulting from insertion mutations in Notch-4 (43%), Wnt-1 (26%), and Fgf-3 (13%). Since the activation of Notch-4 is not common in mammary tumors of standard laboratory strains of mice infected with various MMTV strains, we examined the consequences of Jyg-MMTV infection in BALB/c and C57BL/6 mice. The results show that Jyg-MMTV induces mammary tumors in both mouse strains, but the incidence of mammary tumors in BALB/c mice is greater than in C57BL/6 mice. Surprisingly, however, none of the 75 mammary tumors, analyzed both by Southern and Northern hybridizations, showed insertion mutations in or expression of Notch-4. In contrast, both Wnt-1 and Fgf-3 were found to be involved in these tumors. Our findings may suggest, among other possibilities, the existence of a structural difference(s) between laboratory and wild mice at the Notch-4 locus that regulates the integration of Jyg-MMTV proviral DNA. [Copyright &y& Elsevier]

Abstract: Feline leukemia virus (FeLV) is a gamma retrovirus that induces fatal diseases in domestic cats. Efficacious FeLV vaccines prevent persistent viremia and development of FeLV-related disease after virus exposure, but not minimal viral replication and a provirus-positive state as recently demonstrated using sensitive real-time PCR assays. Proviral integration is an important parameter of latent infection and persistence of retroviruses in infected cells. So far, FeLV-specific real-time PCR assays could not distinguish between the integrated and episomal forms of the provirus. Thus, it was the aim of the present study to develop a rapid assay for the detection of FeLV proviral integration. The test combines conventional and quantitative real-time PCR that use virus-specific primers and primers specific for cat genomic small interspersed nuclear elements. It was applied to analyze the time course of proviral integration into the genome of a feline fibroblast cell line and detect provirus integration in peripheral white blood cells from vaccinated and unvaccinated, FeLV-exposed cats. The newly developed rapid test will essentially contribute to a better understanding of the mechanisms involved in the pathogenesis of FeLV infection and be especially useful in the development of antiretroviral vaccines and therapies aimed at the inhibition of proviral integration. [Copyright &y& Elsevier]

HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells. [Copyright &y& Elsevier]

The pig (Sus scrofa) is a potential organ donor for man but porcine endogenous retroviruses (PERVs) represent an important concern for patients, and identification or engineering of PERV-free pigs suitable for xenotransplantation is a major undertaking. Consequently, studies of variability in pigs for the presence of PERVs at specific loci are a prerequisite. We identified genomic flanking sequences of two PERVs cloned in bacterial artificial chromosomes, a replication-competent PERV-A at locus 1q2.4 and a defective PERV-B at locus 7p1.1–2. PERV-A is embedded in the second repeat of a tandem of eight 190 bp repeats. A short duplicated 4 bp cellular motif, AGAC, was found at each flank of PERV-A and a degenerate 4 bp motif was found for PERV-B. At each locus, the PERV flanks matched expressed sequence tags available in public databases. Primer pairs were designed to amplify either genomic flanks or PERV-genomic junctions. Polymerase chain reaction screening was performed on pigs from 11 distinct Chinese breeds and from the European Large White breed. PERV-B at locus 7p1.1–2 was detected in all animals whereas the presence of PERV-A at locus 1q2.4 was variable. Our results suggest that a genetic selection can be designed to identify animals lacking a potentially active PERV at a specific locus and that Chinese and European pig breeds represent large biodiversity reservoirs to explore. Our results point also to the existence of PERVs that might be fixed in the pig genome, and that might not be eliminated by classical genetic selection. [ABSTRACT FROM AUTHOR]

Integration of viral DNA into the host genome is a central event in the replication cycle and the pathogenesis of retroviruses, including HIV. Although most cells infected with HIV are rapidly eliminated in vivo, HIV also infects long-lived cells that persist during combination antiretroviral therapy (cART). Cells with replication competent HIV proviruses form a reservoir that persists despite cART and such reservoirs are at the center of efforts to eradicate or control infection without cART. The mechanisms of persistence of these chronically infected long-lived cells is uncertain, but recent research has demonstrated that the presence of the HIV provirus has enduring effects on infected cells. Cells with integrated proviruses may persist for many years, undergo clonal expansion, and produce replication competent HIV. Even proviruses with defective genomes can produce HIV RNA and may contribute to ongoing HIV pathogenesis. New analyses of HIV infected cells suggest that over time on cART, there is a shift in the composition of the population of HIV infected cells, with the infected cells that persist over prolonged periods having proviruses integrated in genes associated with regulation of cell growth. In several cases, strong evidence indicates the presence of the provirus in specific genes may determine persistence, proliferation, or both. These data have raised the intriguing possibility that after cART is introduced, a selection process enriches for cells with proviruses integrated in genes associated with cell growth regulation. The dynamic nature of populations of cells infected with HIV during cART is not well understood, but is likely to have a profound influence on the composition of the HIV reservoir with critical consequences for HIV eradication and control strategies. As such, integration studies will shed light on understanding viral persistence and inform eradication and control strategies. Here we review the process of HIV integration, the role that integration plays in persistence, clonal expansion of the HIV reservoir, and highlight current challenges and outstanding questions for future research. [ABSTRACT FROM AUTHOR]