HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE COPIES VERYSHORT TEMPLATES - KINETIC AND CROSS-LINKING ANALYSIS

We describe in this article some properties concerning the cDNA elonga tion activity of human immunodeficiency virus type-1 (HIV-1) reverse t ranscriptase (RT). The kinetic parameters of the polymerization reacti on catalyzed by HIV-1 RT, using short templates, were studied. Values of K(m) and V(max) were measured as a function of the oligoadenylate t emplate length: the logarithm of K(m) increased linearly, with an incr emental factor of 2.2, when the template length differs by one nucleot ide. Using short templates, oligo(A)n (n = 7-14) and primers shorter o r longer than the template, HIV-1 reverse transcriptase was able to sy nthesize polymer products longer than 200 nucleotides. We showed that an oligonucleotide as short as (pA)3 was long enough to serve as templ ate for cDNA synthesis by RT. In the binding of RT to templates of dif ferent lengths (5 to 14 nucleotides long), two constants were determin ed differing in each case by a factor of about 10. The three recombina nt forms of HIV-1 RT (p66/p51, p66/p66 and p51/p51) were crosslinked t o a short template, (pA)14, in the presence of cis-aquahydroxydiammino platinum. The efficiency of crosslink of [P-32](pA)14 template with ea ch of the subunits of RT correlated well with the affinity of this tem plate to the different forms of RT. In the case of p66/p51, the crossl ink occurred mainly with the p66 subunit. These results confirm the im portant catalytic role of the p66 subunit in the heterodimeric human r etroviral polymerase.