In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

We recently reported identification of a previously undescribed gammaretrovirus genome, xenotropic murine leukemia virusrelated virus (XMRV), in prostate cancer tissue from patients homozygous for a reduced activity variant of the antiviral enzyme RNase L. Here we constructed a full-length XMRV genome from prostate tissue RNA and showed that the molecular viral clone is replication-competent. XMRV replication in the prostate cancer cell line DU145 was sensitive to inhibition by IFN-␤. However, LNCaP prostate cancer cells, which are deficient in JAK1 and RNase L, were resistant to the effects of IFN-␤ against XMRV. Furthermore, DU145 cells rendered deficient in RNase L with siRNA were partially resistant to IFN inhibition of XMRV. Expression in hamster cells of the xenotropic and polytropic retrovirus receptor 1 allowed these cells to be infected by XMRV. XMRV provirus integration sites were mapped in DNA isolated from human prostate tumor tissue to genes for two transcription factors (NFATc3 and CREB5) and to a gene encoding a suppressor of androgen receptor transactivation (APPBP2/PAT1/ARA67). Our studies demonstrate that XMRV is a virus that has infected humans and is susceptible to inhibition by IFN and its downstream effector, RNase L.cancer ͉ RNase L ͉ xenotropic murine leukemia virus-related virus A diverse range of mammalian species are susceptible to infections by viruses from the gammaretrovirus genus of Retroviridae (1). Examples of these simple viruses whose genomes include gag, pro, pol, and env genes only are murine leukemia virus (MLV), feline leukemia virus, koala retrovirus, and gibbon ape leukemia virus. These viruses are responsible for leukemogenesis and other diseases in their respective host species (1-3). However, until recently evidence of authentic infections of humans by gammaretroviruses was lacking. We reported in 2006 identification of viral genomes for a previously undescribed gammaretrovirus, termed xenotropic MLV-related virus (XMRV), in a subset of men with prostate cancer (4). The discovery of XMRV followed investigations of the role of the antiviral enzyme RNase L in hereditary prostate cancer, a disease in which tumors arise in three or more first-degree relatives (5). The human RNase L gene (RNASEL) was initially proposed as a candidate for the hereditary prostate cancer 1 (HPC1) gene based on a positional cloning/candidate gene method (6).RNase L is a regulated endoribonuclease for single-stranded RNA that functions in the IFN antiviral response (7,8). IFN treatment of cells induces a family of 2Ј-5Ј oligoadenylate synthetases that produce 5Ј-phosphorylated, 2Ј-5Ј-linked oligoadenylates (2-5A) from ATP in response to stimulation by viral dsRNA. 2-5A activates the preexisting, latent, and ubiquitous RNase L, resulting in degradation of viral and cellular RNA. Sustained activation of RNase L leads to apoptosis, a function consistent with a role in the suppression of tumor growth (9). Although mice lacking RNase L do not spontaneously develop tumors at higher rates than wild-typ...

HIV-1 replication depends on the viral enzyme integrase that mediates integration of a DNA copy of the virus into the host cell genome. This enzyme represents a novel target to which antiviral agents might be directed. Three compounds, 3,5-dicaffeoylquinic acid, 1-methoxyoxalyl-3,5-dicaffeoylquinic acid, and L-chicoric acid, inhibit HIV-1 integrase in biochemical assays at concentrations ranging from 0.06-0.66 microgram/ml; furthermore, these compounds inhibit HIV-1 replication in tissue culture at 1-4 microgram/ml. The toxic concentrations of these compounds are fully 100-fold greater than their antiviral concentrations. These compounds represent a potentially important new class of antiviral agents that may contribute to our understanding of the molecular mechanisms of viral integration. Thus, the dicaffeoylquinic acids are promising leads to new anti-HIV therapeutics and offer a significant advance in the search for new HIV enzyme targets as they are both specific for HIV-1 integrase and active against HIV-1 in tissue culture.

Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4؉ -T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT.