Activity of HIV-1 integrases recovered from subjects with varied rates of disease progression.

We recently described 102 HIV-1 integrase sequences that were amplified from blood cells or plasma obtained up to 18 years ago from 5 hemophiliacs who later died of AIDS and 5 hemophiliacs subsequently classified as slow or nonprogressors ( J Acquir Immune Defic Syndr Hum Retrovirol 1998;19:99-110). Although the region of the HIV-1 genome that encodes integrase was highly conserved, none of the deduced protein sequences of the patient-derived enzymes matched that of the clade B consensus or standard laboratory integrases. To test the hypothesis that the activity of HIV-1 integrases prevalent within an infected person contributes to the rate of disease progression, we have now expressed and purified these proteins and compared them in various assays. Most of the 75 unique full-length integrase proteins from the 102 clones were enzymatically active. Comparison of proteins derived from samples obtained soon after infection showed that the specificity and extent of viral DNA processing and the amount of DNA joining (the two biologically relevant activities of integrase) did not differ between the two groups of patients. In addition, the relative usage of alternative nucleophiles for processing and the amount of nonspecific nicking catalyzed by the proteins were indistinguishable between the patient groups. Although the patient-derived enzymes often exhibited different patterns of target site preferences compared with the laboratory integrase, there was no correlation with clinical course. Thus, the activities of HIV-1 integrases prevalent within these infected individuals, at least as reflected by standard assays, did not influence or predict the rate of disease progression.

Use of patient-derived human immunodeficiency virus type 1 integrases to identify a protein residue that affects target site selection.

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn(2+) or with Mg(2+) and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.

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.

Analysis of a large collection of natural HIV-1 integrase sequences, including those from long-term nonprogressors.

A large collection of natural HIV-1 integrase (IN) sequences has not previously been described. We reasoned that analysis of such sequences would address whether natural variation of HIV-1 IN contributes to the pathogenesis of AIDS and might also identify amino acid residues important for IN function. Sequences encoding HIV-1 IN were amplified from cryopreserved lymphocytes or plasma obtained at different times from 10 hemophilia patients who had been observed for up to 17 years. The region of the HIV-1 genome that encodes the 288-amino acid IN protein was sequenced from a total of 102 clones; information was obtained for 99.97% of 29,478 amino acid positions. Phylogenetic analysis indicated that patient samples were unique. Interpatient nucleic acid distances ranged from 0.8% to 4.9%, highlighting the tight conservation of this genomic region. No major differences were found between DNA and RNA or between early and late time points from the same patient. Significantly, no amino acid changes that might account for the variable rate of disease progression between patients were evident. Only one amino acid substitution involved a highly conserved residue known to be important for enzymatic activity. However, several interesting amino acid substitutions were noted, including residues within the C-terminal region of the protein for which sequence comparisons between animal retroviruses have not been very informative. These results should encourage the pursuit of anti-integrase therapies, especially inasmuch as the apparent biologic constraints on the IN sequence may deter the development of drug resistance.

Inhibition of prokaryotic translation by the Shiga toxin enzymatic subunit.

Shiga toxin, produced by Shigella dysenteriae serotype 1, is a member of the large family of ribosome-inactivating proteins (RIPs) which are primarily produced by plants. All RIPs are rRNA N-glycosidases which inactivate ribosomes through the removal of a specific adenine residue from the well-conserved aminoacyl-tRNA-accepting loop of rRNA. As a type II RIP, STX is believed to have little effect on prokaryotic ribosomes. However, we have demonstrated that over-expression of the STX enzymatic (A1) polypeptide which lacks a signal sequence caused a reduced rate of growth of its Escherichia coli host. Over-expression of the same StxA1 polypeptide with a catalytic site substitution had no effect on the growth of E. coli. In addition, purified StxA1 was an inhibitor of prokaryotic protein synthesis as assessed using an in vitro transcription and translation assay. The specific activity of StxA1 was significantly higher than ricin, which is another type II RIP, with both eukaryotic and prokaryotic translation systems.

Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis.

The enzymatic subunit of Shiga toxin (StxA1) is a member of the ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.