Mutations conferring drug resistance affect eukaryotic expression of HIV type 1 reverse transcriptase.
Mutations in reverse transcriptase (RT) confer high levels of HIV resistance to drugs. However, while conferring drug resistance, they can lower viral replication capacity (fitness). The molecular mechanisms behind remain largely unknown. The aim of the study was to characterize the effect of drug-resistance mutations on HIV RT expression. Genes encoding AZT-resistant RTs with single or combined mutations D67N, K70R, T215F, and K219Q, and RTs derived from drug-resistant HIV-1 strains were designed and expressed in a variety of eukaryotic cells. Expression in transiently transfected cells was assessed by Western blotting and immunofluorescent staining with RT-specific antibodies. To compare the levels of expression, mutated RT genes were microinjected into the nucleus of the oocytes of Xenopus laevis. Expression of RT was quantified by sandwich ELISA. Relative stability of RTs was assessed by pulse-chase experiments. Xenopus oocytes microinjected with the genes expressed 2-50 pg of RT mutants per cell. The level of RT expression decreased with accumulation of drug-resistance mutations. Pulse-chase experiments demonstrated that poor expression of DR-RTs was due to proteolytic instability. Instability could be attributed to additional cleavage sites predicted to appear in the vicinity of resistance mutations. Accumulation of drug-resistance mutations appears to affect the level of eukaryotic expression of HIV-1 RT by inducing proteolytic instability. Low RT levels might be one of the determinants of impaired replication fitness of drug-resistant HIV-1 strains.
Isaguliants
Maria G
MG
Ivanovsky Institute of Virology, 123098 Moscow, Russia. maria.isaguliants@smi.ki.se
Belikov
Sergey V
SV
Starodubova
Elizaveta S
ES
Gizatullin
Rinat Z
RZ
Rollman
Erik
E
Zuber
Bartek
B
Zuber
Anne Kjerrström
AK
Grishchenko
Olga I Andreeva
OI
Rytting
Ann-Sofie
AS
Källander
Clas F R
CF
Kochetkov
Sergey N
SN
Karpov
Vadim L
VL
Wahren
Britta
B
D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D002460:Cell Line; D024882:Drug Resistance, Viral / Q000235:genetics; D004795:Enzyme Stability; D005260:Female; D015870:Gene Expression; D005814:Genes, Viral; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D009865:Oocytes / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis; D015215:Zidovudine / Q000494:pharmacology
D019380:Anti-HIV Agents; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D054303:HIV Reverse Transcriptase