Fig 1.
Schematic representation of the In-fusion cloning steps for the generation of (A) Simian Human Immunodeficiency Virus (SHIV) and (B) Simian tropic Human Immunodeficiency Virus (stHIV). Inverese PCR was used for the linearization of the vector backbone, pSHIVAD8-EO. HIV env gene specific primers were used for the amplification of the each env from specific HIV infectious molecular clones as described previously.
Fig 2.
Genomic organization depicting portions of env used in the construction of (A) SHIVs with full length clade C HIV Zambian T/F env genes (B) SHIVs with truncated env genes and (C) stHIV, where vif of HIV molecular clones were replaced by SIV mac239 vif.
Fig 3.
Infectivity of various SHIVs were measured using β-gal assay.
TZMbl cells were infected with 200TCID50 of 293T cell generated viral supernatant using standard TZMbl assays as described previously. After 72-hrs post infection β-gal assay was measured and Infectious units are calculated. Full-length SHIVs env 375S (panel A) with env 375H (panel C) and env 375Y (panel E). Similarly, truncated SHIVs env 375S (panel B) with env 375H (panel D) and env 375Y (panel F).
Fig 4.
Replication kinetics of newly generated SHIVs in rhesus macaque PBMCs.
Pooled PBMC from randomly selected naive RM donors were stimulated with concanavalin A (Con-A) for 48-72hrs washed and infected with 200TCID50 of various SHIVs as described in the method. Culture supernatents were harvested every three days upto 20-25 days and SIV gag protein (P27) were measured using P27 ELISA kits. Left panel represents a replication kinetics of full-length SHIVs as well as S375H/Y mutation and right panel represents truncated SHIVs and S375H/Y mutation.
Fig 5.
Infectivity of various stHIVs were measured in TZmbl cells using β-gal and luciferase assays.
HEK 293T cells supernatant of newly generated stHIV with Env375H or Env 375Y viruses were infected (200TCID50) with TZMbl cells and after 48-72 hr (A) Infectious units were calculated by β-gal staining to confirm the infectivity of the viruses (B and C) Luciferase activity was measured after adding britelite plus reagent.
Fig 6.
Virus entry assay in ZB5 cells.
Selected full-length and truncated SHIVs with either Env 375S (wt with serine) or Env 375H (mutated to Histidine) or Env 375Y (mutated to Tyrosine) viruses were tested for their ability to enter cells using ZB5 cells which expresses rhesus CD4 and CCR5 at equal level to human CD4 and CCR5 in TZMbl cells. Cells were seeded at 1 × 104 per well in 96-well plates and incubated 200TCID50 of virus for 5 hrs and virus was removed washed cells and replaced with fresh complete medium. After 48 hrs of post-infection britelite plus reagent was added to measure luciferase activity. As a control cells alone without infection were used to correct for background luciferase activity.