Infidelity of DNA synthesis by human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) is a presumptive determinant of HIV-1 hypervariability and is incompletely understood at the mechanistic and structural levels. Amino acid substitution at only three residues, including Asp-76 (Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) Biochemistry 37, 5831-5839), is known to increase fidelity. We report here that substitution at Arg-78 can also increase accuracy. Mutant R78A RT showed reduced primer extension in misincorporation assays lacking a complementary dNTP and exhibited a 9-fold decrease in mutation frequency in the M13mp2 lacZ forward mutation assay. Previous structural studies indicate that Arg-78 and Asp-76 lie in a region that interacts with template nucleotides. Interestingly, R78A RT exhibited 6-to 8-fold decreases in binding affinity (K d ) for RNA and DNA templates relative to wild type RT. In contrast, D76V RT, which also increases fidelity (Kim et al., 1996), showed a 6-to 7-fold increased affinity. The processivity of R78A RT on both RNA and DNA templates was substantially reduced relative to wild type RT, whereas the processivity of D76V RT was increased. We discuss relationships of fidelity, template binding, and processivity in these and other HIV RT mutants.Genomic hypervariability is a defense device that allows human immunodeficiency virus, type 1 (HIV-1) 1 to escape from selection pressure such as that imposed by the host immune system or drug therapies. Hypervariation of HIV-1 genomes can be achieved by at least three unique viral replication processes. First, mutations can be synthesized by the virally encoded DNA polymerase, HIV-1 reverse transcriptase (RT) (1, 2). HIV-1 RT is the most error prone of all known DNA polymerases (3-7). Second, mutations produced by HIV-1 RT can be amplified by recombination, which has been observed in virions containing heterogeneous diploid genomes (8). It has been suggested that the occurrence of homologous recombination during viral replication is related to the processivity and strand transfer activity of HIV-1 RT (9, 10). Finally, constant, massive replication amplifies the complexity of the viral population (11).The structural basis of HIV-1 RT infidelity remains to be fully elucidated. Lack of a 3Ј-5Ј proofreading exonuclease activity is a presumptive factor (4), but it cannot be the only determinant, because other retroviral RTs that lack a proofreading exonuclease, such as murine leukemia virus RT and AMV RT, have 10-to 18-fold higher fidelity (3). Our knowledge of the structure-function relationships governing the accuracy of other DNA polymerases rests heavily on the analysis of mutants (12, 13); the classic mutants of Escherichia coli DNA polymerase I are important examples (14). Currently available mutants of HIV-1 RT are mostly drug-resistant variants isolated from patients and cell culture systems. Interestingly, it has been reported that drug resistance mutations at Met-184 (M184V, M184I) and Glu-89 (E89G) confer increased fidelit...