Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity.
The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.
Molecular Sciences Department, Wellcome Research Laboratories, Beckenham, Kent, U.K.
M
M
Tisdale
T
T
Schulze
B A
BA
Larder
K
K
Moelling
D000595:Amino Acid Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016914:Ribonuclease H; D014162:Transfection
D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H