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115 Membrane Permeabilization by the HIV-1 gp41 Cytoplasmic LLP Domains Differs by Subtype
W. Dowling*1, M. Robb1, D. Birx1, and F. McCutchan2
1Walter Reed Army Inst. of Res., Washington, DC, USA and 2Henry M. Jackson Fndn., Rockville, MD, USA
Background: The HIV-1 gp41 cytoplasmic domain contains 2 amphipathic alpha-helices, LLP-1 and LLP-2, previously shown to alter membrane permeability. The LLP domains may cause cytopathic effects in infected cells by disrupting membrane integrity and can perturb the virion envelope, allowing dNTPs into the viral particle for intravirion reverse transcription. However, only subtype B viruses have been evaluated previously. In this study, the gp41 LLP domains from 39 HIV-1 strains representing 8 subtypes or circulating recombinant forms (CRFs) were evaluated for their ability to permeabilize membranes.
Methods: To assay membrane-permeabilizing activity, a system was employed that was previously used to evaluate gp41 fragments from a subtype B strain. DNA fragments encompassing the gp41 LLP domains were PCR-amplified from subtype A, B, C, D, F, G, H, CRF01_AE, or CRF02_AG reference strains. The products were cloned into a bacterial expression plasmid and used to transform E. coli. Positive clones were grown and protein expression was induced by the addition of IPTG to the culture medium. Membrane permeabilizing activity was measured by changes in growth kinetics.
Results: Induction of proteins from strains of subtypes A, B, C, and D caused inhibition of cell growth. This inhibition was attributed to protein-induced membrane perturbation. In contrast, expression of the proteins from subtypes F, G, H, CRF01_AE, or CRF02_AG had no detectable effect on the cells. A 7-amino acid insertion found in certain subtypes and CRF did not correlate with growth inhibition in this assay. No other motifs were observed to differentiate the active strains from the inactive ones.
Conclusions: HIV-1 gp41 LLP domain peptides from different subtypes vary in their ability to permeabilize membranes. These differences could not be correlated with an insertion found in some of the subtypes and CRF. The amino acid sequence or secondary structure motifs that cause a loss of membrane-permeabilizing activity require further investigation to fully understand the gp41 structure/function relationship.
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