Recombinant HIV type 1 gp41 Antigen, Detection [ENV40]

Human immunodeficiency virus (HIV) is membrane-enveloped, and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41, which is a single-pass integral viral membrane protein. The protein contains an similar to 170-residue ectodomain located outside the virus that is important for fusion and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has noncovalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains similar to 500 residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. This study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH, which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers that are in a native oligomeric state that reflects the initial complex with gp120 and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER and nonhelical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion.

Background: Monitoring of drug resistance-related mutations among patients with recent HIV-1 infection offers an opportunity to describe current patterns of transmitted drug resistance (TDR) mutations. Material/Methods: Of 298 individuals newly diagnosed from March 2008 to February 2014 in southern Poland, 47 were deemed to have recent HIV-1 infection by the limiting antigen avidity immunoassay. Proviral DNA was amplified and sequenced in the reverse transcriptase, protease, and gp41 coding regions. Mutations were interpreted according to the Stanford Database algorithm and/or the International Antiviral Society USA guidelines. TDR mutations were defined according to the WHO surveillance list. Results: Among 47 patients with recent HIV-1 infection only 1 (2%) had evidence of TDR mutation. No major resistance mutations were found, but the frequency of strains with >= 1 accessory resistance-associated mutations was high, at 98%. Accessory mutations were present in 11% of reverse transcriptase, 96% of protease, and 27% of gp41 sequences. Mean number of accessory resistance mutations in the reverse transcriptase and protease sequences was higher in viruses with no compensatory mutations in the gp41 HR2 domain than in strains with such mutations (p=0.031). Conclusions: Despite the low prevalence of strains with TDR mutations, the frequency of accessory mutations was considerable, which may reflect the history of drug pressure among transmitters or natural viral genetic diversity, and may be relevant for future clinical outcomes. The accumulation of the accessory resistance mutations within the pol gene may restrict the occurrence of compensatory mutations related to enfuvirtide resistance or vice versa.