Superinfection of defective human immunodeficiency virus type 1 with different subtypes of wild-type virus efficiently produces infectious variants with the initial viral phenotypes by complementation followed by recombination.

Superinfection rates of human immunodeficiency virus type 1 (HIV-1) have increasingly been leading to more variation in HIV-1, as evidenced by the emergence of circulating recombinant forms (CRFs). We recently reported complementation in a persistently replication-defective subtype B-infected cell clone, L-2, by superinfection with CRF15_01B. The L-2 cells continuously produce immature particles due to a one-base insertion at pol protease. Proviruses in the superinfected cells carried both subtypes and produced particles with a mature morphology. In this study, we examined possible recombination following complementation to generate replication-competent variants by using three cell clones prepared from superinfected L-2 cells. The individual clones predominantly expressed the initial subtype B-derived mature Gag proteins. However, the viral particles carried both subtype B with the mutation and wild-type CRF15_01B at pol, suggesting the generation of virions with heterozygous RNAs. Interestingly, with cell-free passages of the progeny, defective particles disappeared, and were replaced with heterogeneous recombinants in the pol region with sequences derived from CRF15_01B that expressed subtype B phenotype. Thus, even a defective form of persistent HIV-1 can become replication-competent through superinfection-mediated complementation followed by recombination. These findings suggest the significance of long-lived infected cells as recipients for superinfection.