SNPs in APOBEC3 cytosine deaminases and their association with Visna/Maedi disease progression.

The Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) genes are able to inhibit the replication of a wide range of exogenous retroviruses, as well as endogenous retroviruses and retrotransposons. Three APOBEC3 genes, named APOBEC3Z1, APOBEC3Z2 and APOBEC3Z3, have been described in sheep. In this work the three genes have been screened in order to identify polymorphisms. No polymorphism was detected for the A3Z2 and A3Z3 genes but 16 SNPs and a 3-bp deletion were found in the A3Z1 gene. A thermoestability prediction analysis was applied to the detected amino acidic SNPs by three different programs. This analysis revealed a number of polymorphisms that could affect the protein stability. The SNPs of the 3'UTR were tested to detect alterations on the predicted microRNA target sites. Two new microRNA target sites were discovered for one of the alleles. Two SNPs were selected for association studies in relation with the retroviral disease Visna/Maedi in Latxa and Assaf sheep breeds. Although association analyses resulted unconclusive, probably due to the unsuitability of the SNP allele frequency distribution of the selected polymorphisms in the analyzed breeds, these genes remain good candidates for association studies.

Application of general formulas for the correction of a lattice-translocation defect in crystals of a lentiviral integrase in complex with LEDGF.

The symmetry inherent to many biological macromolecular assemblies has been implicated in a range of crystal pathologies, including lattice-translocation defects (LTDs). Crystals suffering from classic LTDs contain two lattices that are shifted with respect to each other but nonetheless remain within the length of coherent interference. LTD introduces an undesirable intensity modulation into diffraction data, resulting in scrambled or partially scrambled electron densities. In this report, LTD theory is extended and a new general method for determining defect fractions is developed based on the heights of the non-origin peaks observed in native Patterson maps. The application of this method to crystals of lentiviral integrase in complex with its cofactor, where the observed translocation vector does not equal a small integral fraction of a unit-cell edge, is reported and its general application to all classic LTD cases is predicted.

Structural basis for functional tetramerization of lentiviral integrase.

Experimental evidence suggests that a tetramer of integrase (IN) is the protagonist of the concerted strand transfer reaction, whereby both ends of retroviral DNA are inserted into a host cell chromosome. Herein we present two crystal structures containing the N-terminal and the catalytic core domains of maedi-visna virus IN in complex with the IN binding domain of the common lentiviral integration co-factor LEDGF. The structures reveal that the dimer-of-dimers architecture of the IN tetramer is stabilized by swapping N-terminal domains between the inner pair of monomers poised to execute catalytic function. Comparison of four independent IN tetramers in our crystal structures elucidate the basis for the closure of the highly flexible dimer-dimer interface, allowing us to model how a pair of active sites become situated for concerted integration. Using a range of complementary approaches, we demonstrate that the dimer-dimer interface is essential for HIV-1 IN tetramerization, concerted integration in vitro, and virus infectivity. Our structures moreover highlight adaptable changes at the interfaces of individual IN dimers that allow divergent lentiviruses to utilize a highly-conserved, common integration co-factor.

An amino acid in the central catalytic domain of three retroviral integrases that affects target site selection in nonviral DNA.

Integrase can insert retroviral DNA into almost any site in cellular DNA; however, target site preferences are noted in vitro and in vivo. We recently demonstrated that amino acid 119, in the alpha2 helix of the central domain of the human immunodeficiency virus type 1 integrase, affected the choice of nonviral target DNA sites. We have now extended these findings to the integrases of a nonprimate lentivirus and a more distantly related alpharetrovirus. We found that substitutions at the analogous positions in visna virus integrase and Rous sarcoma virus integrase changed the target site preferences in five assays that monitor insertion into nonviral DNA. Thus, the importance of this protein residue in the selection of nonviral target DNA sites is likely to be a general property of retroviral integrases. Moreover, this amino acid might be part of the cellular DNA binding site on integrase proteins.

Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages.

Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages.

Characterization of chimeric enzymes between caprine arthritis--encephalitis virus, maedi--visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli.

In order to investigate the functions of the three putative lentiviral integrase (IN) protein domains on viral DNA specificity and target site selection, enzymatically active chimeric enzymes were constructed using the three wild-type IN proteins of caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1). The chimeric enzymes were expressed in Escherichia coli, purified by affinity chromatography and analysed in vitro for IN-specific endonuclease and integration activities on various DNA substrates. Of the 21 purified chimeric IN proteins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integration reaction. Analysis of the chimeric IN proteins revealed that the central domain together with the C terminus determines the activity and substrate specificity of the enzyme. The N terminus appears to have no considerable influence. Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the reaction conditions that support optimal enzyme activities of CAEV IN. Also, under the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibited endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments. Thus, the following report presents a detailed characterization of the activities of CAEV IN in vitro as well as the analysis of functional chimeric lentiviral IN proteins.

Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases.

Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we performed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immuno- deficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA. Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.

Mapping target site selection for the non-specific nuclease activities of retroviral integrase.

To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the human immunodeficiency virus type 1 and visna virus integrases was found to be responsible for selecting non-viral target DNA sites when small alcohols provide the attacking nucleophilic OH group during non-specific alcoholysis assays. Testing deletion derivatives of the integrase protein in this assay, which has similarities to the DNA joining reaction that occurs during retroviral integration, defined a smaller central domain that is sufficient for activity. Thus, this core domain likely contains both the host DNA site and the nucleophile site. Surprisingly, the region of integrase responsible for selecting non-viral target DNA sites when the viral DNA end is the attacking nucleophile could not similarly be mapped with the standard oligonucleotide joining assay. We therefore tested the proteins in a more sensitive assay that displays preferred sites of viral DNA insertion in a plasmid DNA target. All 12 chimeras yielded novel patterns compared with the wild-type enzymes in this assay, although local insertion patterns indicated that the central domain plays an important role in target site selection. Together, these data suggest that other protein regions must be involved when the attacking nucleophilic group is provided by viral DNA. Because specific recognition of viral DNA ends was previously mapped to the central domain, two different regions of integrase must interact with retroviral DNA.

Subterminal viral DNA nucleotides as specific recognition signals for human immunodeficiency virus type 1 and visna virus integrases under magnesium-dependent conditions.

Many reports describe the characteristics of susceptible viral DNA substrates to various retroviral integrases during in vitro reactions in which manganese serves as the divalent cation cofactor for site-specific nicking. However, manganese is known to alter the specificity of some endonucleases and magnesium may be the divalent cation used during retroviral integration in vivo. To address these concerns, we identified conditions under which the integrases of human immunodeficiency virus type 1 and visna virus were optimally active with magnesium (the first time such activity was shown for visna virus integrase) and used these conditions to test the susceptibility of a series of oligodeoxynucleotide substrates. The data show that two base pairs immediately internal to the conserved CA dinucleotide near the termini of retroviral DNA are selectively recognized by the two integrases and that the final six base pairs of viral DNA contain sufficient sequence information for specific recognition and cleavage by each enzyme. The results validate the importance of the subterminal viral DNA positions even in the presence of magnesium and identify viral DNA positions that functionally interact with integrase. The data obtained under magnesium-dependent conditions, which were obtained with substrates containing single and multiple base-pair substitutions and two different retroviral integrases, are consistent with those previously obtained with manganese. Thus, the large body of manganese-dependent data identifying terminal viral DNA positions that are important in substrate recognition by various integrases likely reflects interactions that are biologically relevant.

Mapping viral DNA specificity to the central region of integrase by using functional human immunodeficiency virus type 1/visna virus chimeric proteins.

We previously described the construction and analysis of the first set of functional chimeric lentivirus integrases, involving exchange of the N-terminal, central, and C-terminal regions of the human immunodeficiency virus type 1 (HIV-1) and visna virus integrase (IN) proteins. Based on those results, additional HIV-1/visna virus chimeric integrases were designed and purified. Each of the chimeric enzymes was functional in at least one oligonucleotide-based IN assay. Of a total of 12 chimeric IN proteins, 3 exhibit specific viral DNA processing, 9 catalyze insertion of viral DNA ends, 12 can reverse that reaction, and 11 are active for nonspecific alcoholysis. Functional data obtained with the processing assay indicate that the central region of the protein is responsible for viral DNA specificity. Target site selection for nonspecific alcoholysis again mapped to the central domain of IN, confirming our previous data indicating that this region can position nonviral DNA for nucleophilic attack. However, the chimeric proteins created patterns of viral DNA insertion distinct from that of either wild-type IN, suggesting that interactions between regions of IN influence target site selection for viral DNA integration. The results support a new model for the functional organization of IN in which viral DNA initially binds nonspecifically to the C-terminal portion of IN but the catalytic central region of the enzyme has a prominent role both in specific recognition of viral DNA ends and in positioning the host DNA for viral DNA integration.

Influence of subterminal viral DNA nucleotides on differential susceptibility to cleavage by human immunodeficiency virus type 1 and visna virus integrases.

A comparison of the extents of site-specific cleavage of U5 and U3 viral DNA termini by the integrases of human immunodeficiency virus type 1 and visna virus guided the quantitative testing of oligonucleotide substrates containing specific base substitutions. The simultaneous exchange of positions 5 and 6 between U3 substrates switched the patterns of differential susceptibility to the two integrases. The activity of visna virus integrase was more dependent on the identity of position 5 adjacent to the invariant CA bases than on position 6, whereas human immunodeficiency virus type 1 integrase appeared to interact even more critically with position 6. Although the paired natural substrates of most lentiviral integrases match at positions 7 and 8, these bases were not important for susceptibility of U5 substrates. In fact, the final six U5 positions contained all of the sequence information necessary for susceptibility. These results suggest that constraints other than integration influence the terminal inverted repeats of retroviral DNA.

Mimicry of a 21.5 kDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep.

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TLC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogensis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Replication properties of dUTPase-deficient mutants of caprine and ovine lentiviruses.

The virion-associated dUTPase activities of caprine arthritis-encephalitis virus (CAEV) and visna virus were determined by using an assay which measure the actual ability of the dUTPase to prevent the dUTP misincorporations into cDNA during reverse transcription. We showed that the CAEV molecular clone from the Cork isolate was dUTPase defective as a result of a single amino acid substitution. Using this point mutant and deletion mutants of CAEV as well as a deletion mutant of visna virus, we demonstrated that dUTPase-deficient viruses replicate similarly to wild-type viruses in dividing cells but show delayed replication in nondividing primary macrophages.

Mapping domains of retroviral integrase responsible for viral DNA specificity and target site selection by analysis of chimeras between human immunodeficiency virus type 1 and visna virus integrases.

Human immunodeficiency virus type 1 (HIV-1) and visna virus integrases were purified from a bacterial expression system and assayed on oligonucleotide substrates derived from each terminus of human immunodeficiency virus type 1 and visna virus linear DNA. Three differences between the proteins were identified, including levels of specific 3'-end processing, patterns of strand transfer, and target site preferences. To map domains of integrase (IN) responsible for viral DNA specificity and target site selection, we constructed and purified chimeric proteins in which the N-terminal, central, and C-terminal regions of these lentiviral integrases were exchanged. All six chimeric proteins were active for disintegration, demonstrating that the active site in the central region of each chimera maintained a functional conformation. Analysis of endonucleolytic processing activity indicated that the N terminus of IN does not contribute to viral DNA specificity; this function must reside in the central region or C terminus of IN. In the viral DNA integration assay, chimeric proteins gave novel patterns of strand transfer products which did not match that of either wild-type IN. Thus, target site selection with a viral DNA terminus as nucleophile could not be mapped to regions of IN defined by these boundaries and may involve interactions between regions. In contrast, when target site preferences were monitored with a new assay in which glycerol stimulates IN-mediated cleavage of nonviral DNA, chimeras clearly segregated between the two wild-type patterns. Target site selection for this nonspecific alcoholysis activity mapped to the central region of IN. This report represents the first detailed description of functional chimeras between any two retroviral integrases.

Comparative studies of bacterially expressed integrase proteins of caprine arthritis-encephalitis virus, maedi-visna virus and human immunodeficiency virus type 1.

Integrase (IN) proteins mediate an essential step in retroviral life cycles, the integration of reverse-transcribed viral DNA into the host genome. To create tools for direct comparative investigations, hexahistidine-tagged IN proteins of the phylogenetically related lentiviruses caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1) were expressed in Escherichia coli. After purification by affinity chromatography, the active enzymes were compared in vitro for their site-specific cleavage, integration and disintegration activities on cognate and non-cognate oligonucleotide substrates. It was found that CAEV IN and MVV IN catalyse both site-specific cleavage and disintegration with high efficiencies, reduced substrate specificities and similar reaction patterns. Comparisons with the respective activities of HIV-1 IN revealed basic functional similarities as well as considerable differences such as more restricted substrate requirements for site-specific cleavage. On the other hand, all three enzymes catalyse disintegration almost independent of the substrate origin. Furthermore, MVV IN was shown to join oligonucleotides as efficiently as HIV-1 IN, albeit with reduced substrate specificity. In contrast, no detectable strand transfer activities occurred with CAEV IN.

Ovine aortic smooth muscle cells allow the replication of visna-maedi virus in vitro.

Visna-maedi virus induces in sheep an interstitial lung disease characterised by an accumulation of smooth muscle cells (SMC) or myomatosis. Infection by HIV-1 has been recently associated with disorders of the vessel-derived cells: primary pulmonary hypertension, coronary artery disease and smooth muscle tumors in humans. We hypothesized that, besides their regular targets (i.e. macrophages and lymphocytes), lentiviruses could infect smooth muscle cells. Smooth muscle cell cultures derived from ovine aorta were infected with visna-maedi virus strain K1514. The cultured cells were smooth muscle cells as demonstrated by their antigenic expression of alpha-actin and vimentin. The lentiviral infection of the smooth muscle cells was demonstrated by a typical cytopathic effect (syncytia), the expression of virus specific antigens, and the presence of genomic RNA detected by Northern blot analysis and RT PCR. The detection of a reverse transcriptase activity, the presence of viral RNA in supernatants of infected smooth muscle cells detected by RT PCR and their ability to infect ovine permissive fibroblasts demonstrated a productive infection. The ability of smooth muscle cells to be infected by lentiviruses may participate in the pathogenesis of the tissue damage associated with the lentiviruses such as myomatosis in sheep and vascular disease in humans.

In vitro activities of purified visna virus integrase.

Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration. To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein. We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases. In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity). In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.

Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester.

The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15 microliters of sample, is rapid, and is able to detect virus at titres below 10(3)/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection.

Molecular evolution of the human and simian immunodeficiency viruses.

Molecular evolution and phylogeny of different human immunodeficiency virus type 1 (HIV1) strains, of a type 2 (HIV2) strain, and of two simian immunodeficiency viruses (SIVAGM and SIVMAC) have been studied by comparing the nucleotide sequences of the two regions of their pol genes which encode the reverse transcriptase (RT) and endonuclease/integrase (EN). The analyses show that the different HIV 1s form one cluster (HIV1 group) and that the SIVs and HIV2 form another (HIV2 group). When the entire genomes of a HIV1, a HIV2, and the two SIVs were compared, the SIVAGM showed a unique pattern of mutation accumulations; that is, the SIVAGM has accumulated more nonsynonymous changes than synonymous changes in the RT and EN regions after its recent divergence from SIVMAC-142, and, furthermore, it has a deletion of approximately 350 bp in the region between the pol and env genes. The SIVAGM was apparently derived from cell cultures infected with a macaque isolate, SIVMAC-251. The contamination provides an opportunity to measure the maximum rate of evolution in the SIVAGM by comparing its DNA sequence to those of SIVMAC-251 and SIVMAC-142. The analysis shows that the rates are given approximately by (1.95 +/- 1.37) x 10(-3)/site/year for one SIVAGM sequence and (5.18 +/- 2.25) x 10(-3)/site/year for another.