Functional and defective components of avian endogenous virus long terminal repeat enhancer sequences

Oncogenic avian retroviruses, such as Rous sarcoma virus (RSV) and the avian leukosis viruses, contain a strong enhancer in the U3 portion of the proviral long terminal repeat (LTR). The LTRs of a second class of avian retroviruses, the endogenous viruses (ev) lack detectable enhancer activity. By creating ev-RSV hybrid LTRs, we previously demonstrated that, despite the lack of independent enhancer activity in the ev U3 region, ev LTRs contain sequences that are able to functionally replace essential enhancer domains from the RSV enhancer. A hypothesis proposed to explain these data was that ev LTRs contain a partial enhancer that includes sequences necessary but not sufficient for enhancer activity and that these sequences were complemented by RSV enhancer domains present in the original hybrid constructs to generate a functional enhancer. Studies described in this report were designed to define sequences from both the ev and RSV LTRs required to generate this composite enhancer. This was approached by generating additional ev-RSV hybrid LTRs that exchanged defined regions between ev and RSV and by directly testing the requirement for specific motifs by site-directed mutagenesis. Results obtained demonstrate that ev enhancer sequences are present in the same relative location as upstream enhancer sequences from RSV, with which they share limited sequence similarity. In addition, a 67-bp region from the internal portion of the RSV LTR that is required to complement ev enhancer sequences was identified. Finally, data showing that CArG motifs are essential for high-level activity, a finding that has not been previously demonstrated for retroviral LTRs, are presented.     

Highly potent synthetic polyamides, bisdistamycins, and lexitropsins as inhibitors of human immunodeficiency virus type 1 integrase

Alignment of the available human immunodeficiency virus type 1 (HIV-1) viral DNA termini [U5 and U3 long terminal repeats (LTRs)] shows a high degree of conservation and the presence of a stretch of five or six consecutive adenine and thymine (AT) sequences approximately 10 nucleotides away from each LTR end. A series of AT-selective minor-groove binders, including distamycin and bisdistamycins, bisnetropsins, novel lexitropsins, and the classic monomeric DNA binders Hoechst 33258, 4'-diamino-2-phenylindole, pentamidine, berenil, spermine, and spermidine, were tested for their inhibitory activities against HIV-1 integrase (IN). Although netropsin, distamycin, and all other monomeric DNA binders showed weak activities in the range of 50-200 microM, some of the polyamides, bisdistamycins, and lexitropsins were remarkably active at nanomolar concentrations. Bisdistamycins were 200 times less potent when the conserved AAAAT stretch present in the U5 LTR was replaced with GGGGG, consistent with the preferred binding of these drugs to AT sequences. DNase I footprinting of the U5 LTR further demonstrated the selectivity of these bisdistamycins for the conserved AT sequence. The tested compounds were more potent in Mg+2 than in Mn+2 and inhibited IN50-212 deletion mutant in disintegration assays and the formation of IN/DNA complexes. The lexitropsins also were active against HIV-2 IN. Some of the synthetic polyamides exhibited significant antiviral activity. Taken together, these data suggest that selective targeting of the U5 and U3 ends of the HIV-1 LTRs can inhibit IN function. Polyamides might represent new leads for the development of antiviral agents against acquired immune deficiency syndrome.     

Quantitative in vitro assay for human immunodeficiency virus deoxyribonucleic acid integration

An obligatory step of retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the long terminal repeat (LTR) ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of HIV-1 LTRs as substrate and supercoiled pSP65 DNA as the target, we describe an assay that is suitable for the enzymatic analysis of the integration and for testing candidate inhibitors of HIV IN protein.     

Selective amplification of simian immunodeficiency virus genotypes after intrarectal inoculation of rhesus monkeys

Animal models for sexual transmission of human immunodeficiency virus can define the influences of virus type, dose, and route of inoculation on infection and clinical outcome. We used an uncloned simian immunodeficiency virus stock (SIVmac) to inoculate cells in vitro and to inoculate rhesus monkeys by intravenous and intrarectal routes. The distribution of virus genotypes present in each of these infection examples was characterized by DNA sequence analysis of viral long terminal repeats (LTRs). Our analysis of LTR sequences from in vitro and in vivo infections revealed three main genotypes: one genotype was observed only for in vitro infection, and two other genotypes were recovered only from infected animals. By comparing animals inoculated with high intrarectal doses of SIVmac and those inoculated with low doses, we demonstrated that unique subsets of the stock were selected after intrarectal infection. Our findings indicate that minor genotypes present in the stock cross the rectal mucosa and are amplified selectively to become prominent in peripheral blood mononuclear cells from acutely infected animals. Studies with a molecular recombinant of SIV and human immunodeficiency virus type 1 sequences, SHIV, showed that viral LTR sequences do not undergo especially rapid sequence variation or rearrangement after intrarectal inoculation. The mucosal barrier exerts a significant influence on infection and disease progression by reducing the efficiency of SIVmac infection and by permitting distinct, pathogenic genotypes to become established in the host.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

Human-specific integrations of the HERV-K endogenous retrovirus family

Several distinct families of endogenous retrovirus-like sequences (HERVs) exist in the genomes of humans and other primates. One of these families, the HERV-K group, contains members that encode functional proteins and that have been implicated in the etiology of insulin-dependent diabetes mellitus (IDDM). Because of potential functional and disease relevance, it is important to determine if there are HERV-K-associated genetic differences between individuals. In this study, we have investigated the divergence and evolutionary age of HERV-K long terminal repeats (LTRs). Thirty-seven LTRs, taken primarily from random human clones in GenBank, were aligned and grouped into nine clusters with decreasing sequence divergence. Cluster 1 sequences are 8.6% divergent, on average, whereas cluster 9 LTRs, represented by the LTRs of the fully sequenced HERV-K10 clone, show an average of only 1.1% divergence from each other. The evolutionary age of 18 LTRs from different clusters was then investigated by genomic PCR to determine presence or absence of the retroviral element in different primate species. LTRs from clusters of higher divergence were detected in monkeys and apes, whereas LTRs in clusters with lower divergence were acquired later in evolution. Notably, LTRs of cluster 9 were found only in humans at all nine loci examined. Genomic Southern analysis with an oligonucleotide probe specific for cluster 9 LTRs suggests that HERV-K elements with this type of LTR expanded independently in the genomes of humans and the great apes. This is the first report of endogenous retroviral integrations that are specific to humans and indicates that some HERVs have amplified much later than previously thought. These elements may still be actively transposing and may therefore represent a source of genetic variation linked to disease development.     

Alternate strand DNA triple helix-mediated inhibition of HIV-1 U5 long terminal repeat integration in vitro

Integration of the human immunodeficiency virus (HIV) DNA into the host genome is an obligatory process in the replicative life cycle of the virus. This event is mediated in vitro by integrase, a viral protein which binds to specific sequences located on both extremities of the DNA long terminal repeats (LTRs). These sites are highly conserved in all HIV genomes and thus provide potential targets for the selective inhibition of integration. The integrase-binding site located on the HIV-1 U5 LTR end contains two adjacent purine tracts on opposite strands, 5' . . . GGAAAATCTCT-3'/3'-CCTTTTAGAGA . . . 5', in parallel orientations. A single strand oligonucleotide 5'-GGTTTTTGTGT-3' was designed to associate with these tracts via its ability to form a continuous alternate strand DNA triplex. Under neutral pH and physiological temperature, the oligonucleotide, tagged with an intercalator chromophore oxazolopyridocarbazole, formed a stable triplex with the target DNA. The occurrence of this unusual triplex was demonstrated by both DNase I footprinting and electron microscopy. The triplex inhibits the two steps of the integrase-mediated reactions, namely, the endonucleolytic cleavage of the dinucleotide 5'-GT-3' from the 3' end of the integration substrate and the integration of the substrate into the heterologous target DNA. The midpoints for both inhibition reactions were observed at oligonucleotide concentrations of 50-100 nM. We believe that these results open new possibilities for the specific targeting of viral DNA LTR ends with the view of inhibiting integration under physiological conditions.