Background
The Brec1 recombinase is a genomic engineering approach that shows promise in HIV cure, with in vitro and mouse studies showing efficient excision of HIV provirus. Brec1 recognizes a highly conserved sequence (loxBTR) found in the R region of the long terminal repeats (LTRs) that flank the integrated HIV-1 provirus. The loxBTR sequence is found in more than 90% of group M HIV-1, but is not encoded in the SIV strains used in the nonhuman primate (NHP) model of HIV. Here, we design and characterize 3 distinct chimeric SIV/HIV viruses (SHIVs) encoding loxBTR to enable nonhuman primate (NHP) studies of Brec1.
Methods
Molecular cloning was used to incorporate loxBTR in three ways into a previously validated SHIV infectious molecular clone (IMC), SHIV.C.CH505.v2: by replacing R (Replace-R), replacing the polyA stemloop within R (Replace-PolyA), or inserting loxBTR in U3 (U3-Insertion). loxBTR-IMCs were transfected and assessed for both virion content and infectivity compared to the parental SHIV.C.CH505.v2. Rhesus CD4+ T cells (rhCD4s) were infected and comparative growth kinetics over 15-45 days were captured measuring viral p27. Viral RNA harvested from culture supernatants were sequenced to assess for virus adaptation.
Results
All loxBTR-IMCs generated infectious virions. Replace-PolyA and U3-Insertion demonstrated substantial infectious particle production (43% and 52% of parental IU/µL, respectively), and viral fitness (66% – 75% of parental at day 12, respectively), whereas Replace-R showed minimal virion production and replication (2% and 4% at day 12, respectively). Long-term 45-day cultures led to peak p27 levels of 252ng (U3-Insertion), 212ng (Replace-PolyA), and 143ng (Replace-R) at day 42; no viruses demonstrated adaptation within the loxBTR sites.
Conclusions
loxBTR-SHIVs produce infectious, replication-competent viruses capable of retaining the Brec1 target site over many rounds of productive infection. To our knowledge, this is the first example of a primate lentivirus that is HIV/SIV chimeric in the essential, polyfunctional regulatory region of R. loxBTR-SHIVs offer a new platform for NHP studies investigating Brec1 recombinase-based HIV-1 cure strategies.
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