Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase.

Abstract

We have purified and determined functional parameters of reconstituted, recombinant HIV‐1 reverse transcriptase (RT) heterodimers within which either the p66 or p51 polypeptide was selectively mutated in one or both aspartic acid residues constituting the proposed polymerase active site (‐Y‐M‐D‐D‐). Heterodimers containing a mutated p51 polypeptide retain almost wild type levels of both RNA‐dependent DNA polymerase and ribonuclease H (RNaseH) activity. In contrast, heterodimers whose p66 polypeptide was likewise mutated exhibit wild type RNaseH activity but are deficient in RNA‐dependent DNA polymerase activity. These results indicate that in heterodimer RT, the p51 component cannot compensate for active site mutations eliminating the activity of p66, indirectly implying that solely the p66 aspartic acid residues of heterodimer are crucial for catalysis.

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

“…From the results obtained, we conclude that the ␤ and Pol subunits fulfill mainly a structural function similar to that of the p51 subunits of HIV-1 RT and EIAV RT (13,14,19,21,29,32). However, we cannot completely exclude the possibility that the large subunit fulfills some minor function in catalysis, since mutations in Asp 181 or Asp 505 of the ␤ subunit appear to have some impact on the polymerase or RNase H activities.…”

“…We have shown previously that the different forms of RSV RT can be expressed and purified from insect cells using the baculovirus expression system (37). In order to examine the subunit organization of RSV RT, the technique of subunit-selective mutagenesis was used (13,21) to analyze the effect of RSV RTs carrying a mutation in only one of the two subunits constituting the ␣␤ or ␣Pol heterodimer.…”

“…The polymerase active site is present only in the larger p66 subunit of the heterodimer (13,14,19,36), while the p51 subunit, which is identical in sequence to p66 but lacks the RNase H domain, is not directly involved in catalysis (21). However, p51 has to fulfill an important stabilizing function since the monomeric subunits are inactive (26,27).…”

“…An exchange of the corresponding Asp 185 for His in HIV-1 RT has been shown to drastically reduce the polymerase activity of HIV-1 RT (20). Mutation of this residue to Asn in the p66 subunit of HIV-1 RT yields an enzyme that retains only about 2.1% of the wild-type polymerase activity (21). Kinetic analysis of the mutant HIV-1 RT enzyme revealed a 485-fold reduction in the catalytic constant for dTTP incorporation into a homopolymeric substrate (18).…”

“…We have shown previously that the different forms of RSV RT can be expressed and purified from insect cells using the baculovirus expression system (37). In order to examine the subunit organization of RSV RT, the technique of subunit-selective mutagenesis was used (13,21) to analyze the effect of RSV RTs carrying a mutation in only one of the two subunits constituting the ␣␤ or ␣Pol heterodimer.It has been shown previously by biochemical and crystallographic data that heterodimeric human immunodeficiency virus type 1 (HIV-1) RT p66-p51 reveals an asymmetric subunit organization. The polymerase active site is present only in the larger p66 subunit of the heterodimer (13,14,19,36), while the p51 subunit, which is identical in sequence to p66 but lacks the RNase H domain, is not directly involved in catalysis (21).…”