Effect of Amino Acid Substitutions Within the V3 Region of HIV-1 CRF01_AE on Interaction with CCR5-Coreceptor

Abstract

Specific amino acids within the V3 loop of HIV-1 CRF01_AE envelope glycoprotein that are involved in the interaction with CCR5/CXCR4 coreceptors, are not well characterized. We generated V3 mutants using polymerase chain reaction (PCR)-based site-directed mutagenesis of HIV-1 CRF01_AE R5-env plasmids at specific positions. Mutant viruses were produced by env-pseudotyped virus assay, tested for coreceptor usage using U373.R5 and U373.X4 cells, and viral entry was assessed with luciferase activity measurement. All viruses, harboring either single or double mutations, used the CCR5 coreceptor. However, those containing a single substitution at positions 7, 11, 18, and 32 and those with mutations at positions 5/32 and 18/32 had reduced infectivity. Only virus with arginine substitution at position 11 seemed to be involved in CXCR4 coreceptor usage. Our results suggest that some V3 positions may be necessary for the binding to coreceptor, but not for the switch of coreceptor usage.

Keywords: CCR5; CRF01_AE; HIV-1; V3; coreceptor usage.

FIG. 1.

V3 amino acid sequences of mutants with (A) single mutation or (B) double mutations. Positions in V3 are identified by the number above the R5 parental (WT) sequence. The dots mean that amino acid is similar to the amino acid present in the R5 parental (WT) sequence. Mutations are identified by the bold and underlined amino acid letter codes. WT, wild type.

FIG. 2.

Effect of amino acid mutations on infectivity. Infectivity is expressed as RLUs detected in U373 cells expressing CCR5 (black bar) and CXCR4 (white bar). The results are presented as the mean ± SEM of triplicate infections. LEA and NL4.3 strains were used as positive controls for CCR5-using and CXCR4-using viruses, respectively. NC was pseudotyped virus defective in env resulting from cotransfection of empty PCI expression vector and pNL4.3Δenv.Luc. *p < .05. NC, negative control (defective virus); PCI, PCI expression vector; RLUs, relative light units.