THE MAJORITY OF CELLS ARE SUPERINFECTED IN A CLONED CELL-LINE THAT PRODUCES HIGH-LEVELS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 STRAIN MN

We have isolated seven single-cell clones from an H9 culture infected with human immunodeficiency virus type 1 strain MN so that a stable pr oducer of virus could be obtained. DNAs from these clones were examine d by Southern blot analysis and found to contain between one and four proviruses per clone. One of these cell lines, Clone 4, produced high levels of replication-competent virus and contained two proviruses. So uthern blot analysis of DNAs from Clone 4 revealed that, after extende d culture, some of the cells had acquired additional proviruses, presu mably by superinfection. Analysis of Clone 4 single-cell subclones iso lated from a late-passage culture found that 14 out of 20 (70%) subclo nes were reinfected and that 8 out of 20 (40%) were reinfected more th an once. Fluorescence-activated cell sorter analysis showed that surfa ce CD4 levels on Clone 4 cells were appropriately down-regulated. Our results indicate that while there is significant interference to super infection in the Clone 4 culture, it is not absolute and that superinf ected cells accumulate in the culture over time in the presence of hig h virus exposure and extensive cell-to-cell contact. Given our data, i t seems likely that superinfection can occur in vivo within the lympho id reservoirs that harbor human immunodeficiency virus type 1 during t he clinically latent period and may contribute to disease progression.