Citation

  • Authors: Moyo, T., Ereno-Orbea, J., Jacob, R. A., Pavillet, C. E., Kariuki, S. M., Tangie, E. N., Julien, J. P., Dorfman, J. R.
  • Year: 2018
  • Journal: J Virol
  • Applications: in vitro / DNA / FectoPRO
  • Cell types:
    1. Name: HEK-293FT
    2. Name: HEK-293S

Method

HEK293F (Thermo Fisher Scientific) or HEK293 Gnt I-/- (HEK293S) (ATCC® CRL-3022™) suspension cells were split in 200 ml cultures at 0.8 x 10^6 cells/ml. 50 μg of DNA was filtered and mixed in a 1:1 ratio with transfection reagent FectoPRO (Polyplus-Transfection). The DNA:FectoPRO solution was incubated with the cells at 37°C. Cells were harvested 6-7 days post-transfection by centrifugation and supernatants were filtered using a 0.22 μm Steritop filter (EMD Millipore).

Abstract

Understanding the mechanisms used by HIV-1 to evade antibody neutralization may contribute to the design of a high-coverage vaccine. The tier 3 virus 253-11, is poorly neutralized by subtype-matched and subtype C sera, even when compared to other tier 3 viruses, and is also recognized poorly by V3/glycan targeting monoclonal antibodies. We found that sequence polymorphism in the V3 loop and N-linked glycosylation sites only minimally contribute to the high neutralization resistance of 253-11. Interestingly, the 253-11 membrane proximal external region (MPER) is rarely recognized by sera in the context of the wild-type virus, but is commonly recognized in the context of an HIV-2 chimeric virus, suggesting steric or kinetic hindrance of binding to MPER in the native Env. Mutations in the 253-11 MPER - which were previously reported to increase the lifetime of the pre-fusion Envelope (Env) conformation - affected the resistance of 253-11 to antibodies targeting various epitopes on HIV-1 Env, presumably destabilizing its otherwise stable, closed trimer structure. To gain insight into the structure of 253-11, we constructed and crystallized a recombinant 253-11 SOSIP trimer. The resulting structure revealed that the heptad repeat helices in gp41 are drawn in close proximity to the trimer axis and that gp120 protomers also showed a relatively compact disposition around the trimer axis. These observations give substantial insight into the molecular features of an envelope spike from a tier 3 virus and into possible mechanisms that may contribute to its unusually high neutralization resistance.IMPORTANCE HIV-1 isolates that are highly resistant to broadly neutralizing antibodies could limit the efficacy of an antibody-based vaccine. We studied 253-11, which is highly resistant to commonly-elicited neutralizing antibodies. To further understand its resistance, we made mutations that are known to delay fusion and thus increase the time the virus spends in the open conformation following CD4-binding. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4 binding, presumably by destabilizing the trimer structure. To gain further information about the structure of the 253-11 Env trimer, we generated a recombinant 253-11 SOSIP trimer. The crystal structure of the SOSIP trimer revealed that the gp41 helices and the gp120 protomers were drawn in towards the center of the molecule compared to most solved HIV-1 Env structures. These observations provide insight into the distinct molecular features of a Tier 3 envelope spike.

Pubmed