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

Abstract

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.

Specific and Independent Recognition of U3 and U5 att Sites by Human Immunodeficiency Virus Type 1 Integrase In Vivo

“…Here again, we observed a tolerance for mutations in the U3 or U5 att region in the MuLV att chimeric virus. Thus, our results clearly demonstrated the att site specificity of HIV-1 IN in vivo, consistent with previous in vitro studies (27,28,35). On the other hand, replacement of the att sequence with FIV att showed less of an effect on integration in vivo.…”

“…1). Importantly, the critical nucleotides in each att region that might determine the substrate specificity of HIV-1 IN, as indicated in the chimeric att mutant experiment and previous in vitro studies (27,56), were not conserved between the two att regions. Thus, it is suggested that IN might recognize U3 and U5 of att independently.…”

“…Importantly, alteration of the conserved CA dinucleotide significantly impairs the in vitro IN activities, i.e., 3Ј processing, strand transfer, and disintegration reactions (8,10,31,35,49,54). In addition to the CA dinucleotide, the importance of the subterminal LTR sequence for specific interaction has also been reported (2,3,27,31,35,49,54,56). However, interpretation of these in vitro assays is limited since they examine the action of IN on only a single viral DNA end.…”

High affinity interaction of HIV‐1 integrase with specific and non‐specific single‐stranded short oligonucleotides

“…Some reports have suggested that there is limited sequence specificity required, with only the well conserved CA dinucleotide being needed for processing and strand transfer [25]. Other studies have suggested the importance of 2–7 base pairs upstream of the conserved CA, with varying effects on the reaction [26–30]. Recently, the importance of residues at positions 8–11 distal to the LTR which interact with the C‐terminal domain of IN has been reported [31].…”

Mapping Viral DNA Specificity to the Central Region of Integrase by Using Functional Human Immunodeficiency Virus Type 1/Visna Virus Chimeric Proteins

“…Our preparations of HIV-1 IN and visna virus IN process approximately 40 and 50% of this substrate, respectively (Fig. 4A, lanes 3 and 4), as determined using a highly reproducible and conservative method to quantify the extent of specific cleavage (37). Thus, assays with the HIV-1 U5 substrate can indicate whether a chimeric protein has any processing activity.…”