Table 2.
env clonal analysis of four patient virus populations containing mixed amino acid substitutions at position 11 in V3
| Subject (subtype) |
Virusesa | Position 11b |
V3 amino acid sequencesc | No.of AA changesd |
Coreceptor tropisme |
Infectivity (RLU)f | |
|---|---|---|---|---|---|---|---|
| CXCR4+ cells |
CCR5+ cells |
||||||
| 1(B/C) | Population | S/R | DM | 14,188 | 523,551 | ||
| clone (n=4) | S |  |
0 | R5 | 83 | 951,905 | |
| clone (n=1) | S | 4 | R5 | 63 | 556,160 | ||
| clone (n=1) | S | 5 | R5 | 157 | 1,015,357 | ||
| clone (n=2) | R | 8 | Dual-X | 564,997 | 432,732 | ||
| 2(B) | Population | G/R | DM | 73,347 | 55,808 | ||
| clone (n=1) | G |  |
0 | R5 | 177 | 69,366 | |
| clone (n=2) | G | 0 | Dual-R | 660 | 1,456,992 | ||
| clone (n=4) | R | 5 | Dual-X | 681,919 | 350,020 | ||
| clone (n=3) | R | 6 | Dual-X | 730,141 | 174,252 | ||
| 3(AE) | Population | S/R | DM | 6,091 | 142,291 | ||
| clone (n=4) | S |  |
0 | R5 | 121 | 603,308 | |
| clone (n=1) | S | 2 | R5 | 74 | 1,211,595 | ||
| clone (n=1) | S | 2 | Dual-X | 16,534 | 13,848 | ||
| clone (n=3) | R | 5 | Dual-X | 1,026,300 | 693,476 | ||
| 4(B) | Population | S/R | DM | 560,500 | 745,641 | ||
| clone (n=6) | S |  |
0 | Dual-X | 879,283 | 531,108 | |
| clone (n=1) | R | 1 | Dual-X | 173,666 | 126,407 | ||
| clone (n=1) | R | 2 | Dual-X | 29,439 | 40,645 | ||
Clones with identical V3 sequences and the same coreceptor tropism are grouped together. One dual clone from subject 2 and one from subject 3 (indicated in bold) were chosen for reverse site directed mutagenesis.
The 11R substitutions in V3 are shown in boldface
For V3 amino acid sequences, dots represent amino acids identical to the first sequence, dashes in the first sequence indicate gaps to accommodate insertions. Positions 11, 25 and 6–8 (for PNGS) are underlined.
The number of amino acids that differ from the most prevalent V3 sequence of R5 clones in the same sample is indicated, except for subject 4. Subject 4 contained only dual clones, and the number of amino acid differences between clones with or without 11R is shown.
Coreceptor tropism was determined by the Trofile assay. Dual clones were further classified as dual-X and dual-R based on efficiently or inefficiently utilized CXCR4, as defined previously (Huang et al., 2007). R5 and dual-X clones from subject 3 that shared identical V3 amino acid sequences are highlighted (bold and italics). Dual-X clones from subject 4 that shared identical V3 amino acid sequences, except at position 11 (S or R), are highlighted (bold and italics).
Infectivity was measured as luciferase activity (relative light units, RLU) in the Trofile assay. RLU <200 on CXCR5+ or CXCR+ cells was considered background luciferase activity in this study. Infectivity of multiple clones with the same V3 sequence is expressed as the average RLU.