Summary of RT activity and western blot analysis of HIV-1 RT mutants.
The following data is an updated version of Table 7 from the Ph.D.
thesis of John A. Wrobel (1996). It is posted as a supplement to
Wrobel, J.A., Chao, S.-F., Conrad, M.J., Merker, J.D., Swanstrom, R.,
Pielak, G.J., and Hutchison, C.A.III (1998). A genetic approach for
identifying critical residues in the fingers and palm subdomains of HIV-
1 reverse transcriptase. Proc. Natl. Acad. Sci. USA 95, 638-645.
RT assay results for each mutant are listed as percentage of wild-
type activity. Mutant RT assay phenotypes are classified as: negative
(-) for <3% of wild-type activity; low intermediate (+) for >3% but <20
of wild-type activity; high intermediate (++) for >20% but <50 of wild-
type activity; and wild-type or positive (+++) for >50% of wild-type
activity. Western blot results were obtained for both total cell
lysates and soluble lysates. The western results (residues P95-
M164) for total cell lysates are classified as wild-type (wt) for the
presence of wild-type levels of the p66 and p51 forms of the mature
RT with no unprocessed high molecular weight bands; upper strong
(US) for the presence of wild-type levels (strong) of the p66 and p51
forms of the mature RT with unprocessed high molecular weight
(upper) bands; upper light (UL) for the presence of reduced levels
(light) of the p66 and p51 forms of the mature RT with unprocessed
high molecular weight (upper) bands; and light (Lt) for the presence
of reduced levels (light) of p66 and p51 forms of the mature RT with
no unprocessed high molecular weight bands. The western results
(M164-E203) for total cell lysates are classified as wild-type or
positive (wt): efficient processing to produce stable products, as
indicated by the presence of wild-type levels of p66 and p51 forms
of the mature RT; negative (neg): inefficient processing, indicated by
the near absence of p51, a much reduced level of p66, with some
high and low molecular weight products when compared to a wild-
type clone; and intermediate (I): this category contains the widest
variety of phenotypes, generally indicated by a reduced level of the
RT heterodimer. The western results for soluble lysates are
classified as wild-type (wt) for the presence of wild-type levels of
the p66 and p51 forms of the mature RT; faint for reduced levels of
p66 and p51; very faint for a further reduction of p66 and p51 when
compared to those in the faint category; and no bands for the
complete absence of p66 and p51. Mutants that were not assayed in
soluble lysate westerns are indicated with n.d. (not determined). The
Genetic Classification of the amino acid residues from our mutational
analysis (stability, catalytic, functional, external) uses "UPPER CASE"
to indicate those residues that were assigned a role from a
conservative interpretation of the phenotype data, with additional
residues from a more liberal interpretation indicated by "lower case."
Western Western Genetic
Mutant Change RT assay (total cell) (soluble) Classification
P95A C 5 ( +) wt wt FUNCTIONAL
E NC 1.5 ( -) wt wt FUNCTIONAL
L NC 2.3 ( -) wt wt FUNCTIONAL
H96N NC 60 (+++) wt n.d.
L NC 9 ( +) US n.d.
P97A C 63 (+++) wt n.d. STABILITY
L NC 2 ( -) UL faint STABILITY
A98E NC 5 ( +) Lt very faint
V NC 8 ( +) wt n.d.
P C 2.7 ( -) wt wt
G99A C 14 ( +) wt wt FUNCTIONAL
E NC 0.6 ( -) wt n.d. FUNCTIONAL
L NC 2.2 ( -) US n.d. FUNCTIONAL
T C 2.6 ( -) wt n.d. FUNCTIONAL
L100F NC 12 ( +) wt wt
I C 19 ( +) wt n.d.
Q NC 8 ( +) US wt
K101Q NC 113 (+++) wt n.d. EXTERNAL
R C 85 (+++) wt wt EXTERNAL
K102E NC 41 ( ++) Lt very faint
R C 63 (+++) wt n.d.
M NC 44 ( ++) wt wt
Q NC 114 (+++) wt wt
K103N NC 84 (+++) wt n.d. EXTERNAL
T NC 70 (+++) US wt EXTERNAL
K104T NC 73 (+++) wt n.d. EXTERNAL
S105A C 62 (+++) wt wt
L NC 8 ( +) wt wt
V106I C 91 (+++) wt n.d. EXTERNAL
Q NC 57 (+++) US wt EXTERNAL
T107A C 61 (+++) wt n.d. stability
P C <1 ( -) UL no bands stability
R NC 5 ( +) UL no bands stability
S C 69 (+++) wt n.d. stability
V NC 61 (+++) US wt stability
V108E NC 2 ( -) UL faint stability
G NC 5 ( +) Lt very faint stability
L109G NC <1 ( -) Lt very faint
M C 170 (+++) wt n.d.
P NC 2 ( -) UL no bands
Q NC 1 ( -) UL faint
W NC 2 ( -) wt n.d.
D110G NC 2 ( -) wt n.d. CATALYTIC
N C 1 ( -) wt wt CATALYTIC
V NC <1 ( -) wt wt CATALYTIC
Y NC 2 ( -) wt n.d. CATALYTIC
V111E NC <1 ( -) UL no bands
G NC 2 ( -) wt n.d.
R NC <1 ( -) UL faint
G112A C 91 (+++) wt n.d.
F NC 21 ( ++) US wt
D113E C 43 ( ++) wt n.d.
H NC 31 ( ++) wt n.d.
V NC 7 ( +) US wt
Y NC 5 ( +) wt n.d.
A114G C 85 (+++) wt n.d. FUNCTIONAL
V NC 2 ( -) wt n.d. FUNCTIONAL
Y115F C 52 (+++) wt wt
S NC 6 ( +) wt wt
F116C NC 40 ( ++) wt n.d.
L NC 93 (+++) wt n.d.
V NC 42 ( ++) wt n.d.
S117A C 46 ( ++) US wt FUNCTIONAL
L NC 1.7 ( -) wt wt FUNCTIONAL
V118D NC 2 ( -) UL faint STABILITY
T NC 36 ( ++) wt n.d. STABILITY
I C 104 (+++) wt wt STABILITY
P119H NC 81 (+++) wt n.d.
L NC 34 ( ++) US wt
R NC 53 (+++) wt wt
L120F NC 8 ( +) UL no bands stability
W NC 1 ( -) UL no bands stability
A NC 0.9 ( -) UL very faint stability
T NC 1 ( -) UL very faint stability
D121A NC 68 (+++) wt n.d.
E C 70 (+++) US wt
G NC 32 ( ++) US wt
V NC 103 (+++) US wt
Y NC 135 (+++) wt n.d.
E122A NC 42 ( ++) wt n.d.
D C 64 (+++) wt n.d.
G NC 38 ( ++) US wt
V NC 64 (+++) US wt
D123E C 89 (+++) wt n.d.
N C 66 (+++) wt n.d.
Y NC 39 ( ++) US wt
F124I NC 19 ( +) Lt n.d. stability
L NC 6 ( +) Lt very faint stability
S NC 2 ( -) UL very faint stability
V NC 18 ( +) Lt n.d. stability
R125K C 87 (+++) wt wt STABILITY
W NC 1 ( -) UL no bands STABILITY
K126R C 50 (+++) wt n.d. stability
L NC 17 ( +) UL faint stability
Y127D NC 1 ( -) UL no bands stability
F C 51 (+++) wt n.d. stability
H NC 8 ( +) Lt no bands stability
N NC 8 ( +) Lt very faint stability
T128D NC <1 ( -) UL no bands STABILITY
P C <1 ( -) UL no bands STABILITY
V NC 4 ( +) UL very faint STABILITY
A129E NC <1 ( -) UL no bands STABILITY
G C 26 ( ++) Lt faint STABILITY
L NC 1 ( -) UL no bands STABILITY
F130C NC 2 ( -) UL no bands STABILITY
L NC 2 ( -) UL very faint STABILITY
T131I NC 11 ( +) UL faint stability
P C <1 ( -) Lt no bands stability
S C 8 ( +) US faint stability
I132K NC <1 ( -) UL no bands STABILITY
L C 42 ( ++) wt n.d. STABILITY
R NC 1 ( -) UL very faint STABILITY
T NC 1 ( -) UL very faint STABILITY
P133A C 11 ( +) Lt very faint stability
H NC 4 ( +) Lt very faint stability
L NC 1 ( -) Lt no bands stability
R NC 8 ( +) UL very faint stability
S C 8 ( +) UL very faint stability
T C 7 ( +) UL faint stability
S134C NC 53 (+++) wt n.d.
G C 17 ( +) wt wt
N NC 39 ( ++) wt wt
R NC 40 ( ++) wt wt
Y NC 15 ( +) wt wt
I135V C 95 (+++) wt n.d.
E NC 27 ( ++) wt wt
N136H NC 1 ( -) wt n.d. FUNCTIONAL
I NC 1 ( -) US wt FUNCTIONAL
K NC <1 ( -) wt wt FUNCTIONAL
N137D C 5 ( +) wt n.d.
H NC 38 ( ++) wt n.d.
I NC 7 ( +) US wt
K NC 2 ( -) wt n.d.
E138A NC 67 (+++) wt n.d. EXTERNAL
D C 66 (+++) wt n.d. EXTERNAL
G NC 63 (+++) wt n.d. EXTERNAL
T139I NC 55 (+++) wt n.d.
K NC 88 (+++) wt n.d.
P C 1 ( -) US wt
R NC 72 (+++) wt n.d.
P140A C 12 ( +) wt n.d. FUNCTIONAL
L NC 2 ( -) US wt FUNCTIONAL
R NC 1 ( -) wt n.d. FUNCTIONAL
T C 40 ( ++) wt n.d. FUNCTIONAL
G141E NC 1 ( -) wt wt
R NC 2 ( -) wt n.d.
V NC 4 ( +) wt n.d.
I142F NC 56 (+++) wt n.d. EXTERNAL
M C 76 (+++) wt n.d. EXTERNAL
N NC 51 (+++) wt n.d. EXTERNAL
R143I NC 1 ( -) UL faint STABILITY
S NC 2 ( -) UL faint STABILITY
Y144C NC 2 ( -) UL faint STABILITY
D NC 1 ( -) UL no bands STABILITY
F C 61 (+++) US wt STABILITY
N NC <1 ( -) UL faint STABILITY
Q145E C 88 (+++) wt n.d.
L NC 28 ( ++) US n.d.
Y146C NC 2 ( -) UL no bands STABILITY
D NC 2 ( -) UL no bands STABILITY
F C 34 ( ++) wt wt STABILITY
N NC 1 ( -) UL no bands STABILITY
N147D C 34 ( ++) US wt stability
T NC 19 ( +) Lt very faint stability
V148A NC 6 ( +) Lt very faint stability
L C 2 ( -) UL no bands stability
L149I C 30 ( ++) wt wt STABILITY
P NC 1 ( -) UL very faint STABILITY
R NC 2 ( -) Lt no bands STABILITY
V C 5 ( +) US faint STABILITY
P150L NC 2 ( -) UL no bands STABILITY
Q151H NC 76 (+++) wt n.d.
P NC 24 ( ++) wt wt
R NC 2 ( -) wt n.d.
G152R NC <1 ( -) UL very faint
V NC <1 ( -) wt n.d.
W153C NC 13 ( +) US very faint stability
F C 112 (+++) wt n.d. stability
G NC 4 ( +) UL faint stability
K154E NC 2 ( -) wt n.d.
I NC 18 ( +) wt wt
G155E NC 4 ( +) UL very faint
R NC 1 ( -) wt n.d.
S156L NC 1 ( -) wt n.d. FUNCTIONAL
A C 88 (+++) wt wt FUNCTIONAL
P157R NC <1 ( -) wt n.d. FUNCTIONAL
S C 40 ( ++) wt n.d. FUNCTIONAL
T C 45 ( ++) wt n.d. FUNCTIONAL
A158S C 79 (+++) wt n.d. FUNCTIONAL
T C 46 ( ++) wt n.d. FUNCTIONAL
L NC 2.1 ( -) wt wt FUNCTIONAL
I159K NC 4 ( +) UL very faint stability
M C 17 ( +) Lt faint stability
R NC 1 ( -) Lt no bands stability
F160C NC 10 ( +) wt n.d.
L NC 58 (+++) wt n.d.
Y C 28 ( ++) wt n.d.
Q161E C 75 (+++) wt wt
H NC 6 ( +) wt n.d.
I NC 32 ( ++) wt n.d.
L NC 15 ( +) wt n.d.
P NC 4 ( +) Lt faint
S162C NC 39 ( ++) wt n.d.
N NC 75 (+++) wt n.d.
R NC 30 ( ++) wt n.d.
S163C NC 90 (+++) wt n.d.
I NC 54 (+++) wt n.d.
R NC 17 ( +) wt n.d.
M164I C 68 (+++) wt wt stability
K NC 1 ( -) Lt n.d. stability
L C 116 (+++) wt wt stability
V C 16 ( +) wt n.d. stability
T NC 4 ( +) I n.d. stability
T165S C 146 (+++) wt wt
I NC 48 ( ++) wt wt
K166R C 52 (+++) I n.d.
T NC 41 ( ++) wt n.d.
I167L C 40 ( ++) wt n.d. stability
M C 50 (+++) wt n.d. stability
S NC 2 ( -) I n.d. stability
T NC 4 ( +) neg n.d. stability
L168F NC 2 ( -) I n.d. STABILITY
S NC 1 ( -) neg n.d. STABILITY
E169D C 125 (+++) wt wt stability
V NC 19 ( +) I n.d. stability
P170A C 50 (+++) wt wt
S C 45 ( ++) wt n.d.
H NC 20 ( ++) I n.d.
L NC 25 ( ++) wt n.d.
R NC 16 ( +) I n.d.
F171Y C 56 (+++) wt n.d. stability
C NC 2 ( -) neg n.d. stability
I NC 4 ( +) neg n.d. stability
L NC 7 ( +) I n.d. stability
R172K C 42 ( ++) wt wt
S NC 8 ( +) wt wt
T NC 8 ( +) wt wt
G NC 3 ( +) I n.d.
K173R C 149 (+++) wt wt EXTERNAL
T NC 86 (+++) wt wt EXTERNAL
Q NC 106 (+++) wt wt EXTERNAL
N NC 95 (+++) wt wt EXTERNAL
I NC 98 (+++) wt wt EXTERNAL
Q174E C 133 (+++) wt wt
K NC 95 (+++) wt wt
P NC 5 ( +) I n.d.
R NC 89 (+++) wt wt
L NC 104 (+++) wt wt
N175K NC 1 ( -) I n.d. stability
T NC 5 ( +) I n.d. stability
S NC 8 ( +) I n.d. stability
P176A C 61 (+++) I n.d.
L NC 35 ( ++) I n.d.
R NC 55 (+++) wt n.d.
D177E C 135 (+++) wt wt
H NC 80 (+++) wt wt
V NC 14 ( +) I wt
A NC 71 (+++) wt wt
I178V C 15 ( +) neg n.d.
L C 128 (+++) wt n.d.
T NC 48 ( ++) I n.d.
R NC 2 ( -) neg n.d.
V179D NC 145 (+++) wt n.d.
F NC 4 ( +) I n.d.
I180M C 20 ( ++) I n.d.
L C 54 (+++) wt n.d.
F NC 9 ( +) wt wt
T NC 3 ( +) wt n.d.
S NC <1 ( -) neg n.d.
N NC <1 ( -) neg n.d.
Y181F C 13 ( +) I n.d.
S NC 8 ( +) I n.d.
H NC 15 ( +) wt wt
Q182E C 2 ( -) I n.d.
P NC 1 ( -) I wt
R NC 0 ( -) I n.d.
H NC 24 ( ++) wt wt
K NC 0 ( -) I n.d.
Y183F C 20 ( ++) wt n.d. FUNCTIONAL
S NC <1 ( -) wt wt FUNCTIONAL
N NC 0 ( -) wt wt FUNCTIONAL
C NC 5 ( +) wt wt FUNCTIONAL
D NC 0 ( -) wt n.d. FUNCTIONAL
H NC 2 ( -) wt wt FUNCTIONAL
M184I C 35 ( ++) wt n.d. FUNCTIONAL
L C 25 ( ++) wt wt FUNCTIONAL
V C 45 ( ++) I n.d. FUNCTIONAL
K NC 0 ( -) wt wt FUNCTIONAL
D185E C 1 ( -) wt wt CATALYTIC
N C 0 ( -) wt n.d. CATALYTIC
V NC 0 ( -) I n.d. CATALYTIC
G NC 0 ( -) wt wt CATALYTIC
Y NC 0 ( -) wt n.d. CATALYTIC
D186E C <1 ( -) wt wt CATALYTIC
N C 0 ( -) wt wt CATALYTIC
A NC 0 ( -) wt wt CATALYTIC
H NC 0 ( -) wt wt CATALYTIC
G NC <1 ( -) wt wt CATALYTIC
Y NC 0 ( -) wt wt CATALYTIC
L187V C 14 ( +) wt wt stability
M C 86 (+++) wt wt stability
F NC 9 ( +) I n.d. stability
W NC 0 ( -) I n.d. stability
S NC 0 ( -) neg n.d. stability
Y188F C 43 ( ++) wt wt stability
C NC 13 ( +) I n.d. stability
V189I C 20 ( ++) wt n.d. stability
L C 33 ( ++) I wt stability
A NC 11 ( +) I n.d. stability
G NC 0 ( -) neg n.d. stability
G190A C 75 (+++) wt wt stability
R NC 2 ( -) I n.d. stability
E NC 4 ( +) I n.d. stability
S191A C 2 ( -) neg no bands STABILITY
P C 0 ( -) neg n.d. STABILITY
F NC 0 ( -) neg n.d. STABILITY
Y NC 0 ( -) neg n.d. STABILITY
T C 81 (+++) wt wt STABILITY
D192E C 57 (+++) wt wt
N C 46 ( ++) wt wt
Y NC 6 ( +) I n.d.
V NC 4 ( +) I n.d.
G NC 21 ( ++) I n.d.
A NC 14 ( +) I n.d.
L193I C 42 ( ++) I wt
V C 33 ( ++) I n.d.
F NC 127 (+++) wt wt
E194D C 145 (+++) wt wt EXTERNAL
K NC 95 (+++) wt wt EXTERNAL
G NC 107 (+++) wt wt EXTERNAL
V NC 83 (+++) wt wt EXTERNAL
I195L C 115 (+++) wt wt EXTERNAL
T NC 125 (+++) wt wt EXTERNAL
K NC 96 (+++) wt wt EXTERNAL
R NC 80 (+++) wt wt EXTERNAL
G196V NC 39 ( ++) wt n.d.
W NC 53 (+++) wt n.d.
R NC 28 ( ++) I n.d.
Q197E C 74 (+++) wt wt EXTERNAL
R NC 61 (+++) wt wt EXTERNAL
L NC 52 (+++) wt wt EXTERNAL
H NC 77 (+++) wt wt EXTERNAL
H198R C 1 ( -) neg n.d. STABILITY
Y NC 2 ( -) neg n.d. STABILITY
N NC 1 ( -) neg n.d. STABILITY
P NC 0 ( -) neg n.d. STABILITY
Q NC 14 ( +) I faint STABILITY
R199T NC 37 ( ++) I n.d.
I NC 9 ( +) neg n.d.
S NC 32 ( ++) I n.d.
T200A C 111 (+++) wt wt
S C 65 (+++) wt wt
P C 2 ( -) neg n.d.
I NC 42 ( ++) I wt
K NC 51 (+++) wt n.d.
R NC 124 (+++) wt wt
K201N NC 5 ( +) neg n.d.
I NC 53 (+++) wt n.d.
I202V C 108 (+++) wt wt STABILITY
M C 21 ( ++) wt wt STABILITY
K NC <1 ( -) neg n.d. STABILITY
R NC <1 ( -) neg n.d. STABILITY
E203D C 89 (+++) wt wt
V NC 58 (+++) wt wt
G NC 22 ( ++) I n.d.
A NC 55 (+++) wt n.d.