Human immunodeficiency virus type 1 subtypes have a distinct long terminal repeat that determines the replication rate in a host-cell-specific manner

Abstract

The long terminal repeat (LTR) transcriptional promoters of different human immunodeficiency virus (HIV) type 1 subtypes were inserted into the LAI molecular clone of subtype B. The viral genotypes represent seven subtypes (A, B, C, D, E, F, and G) and one circulating recombinant form (AG). We performed replication studies with this isogenic set of viruses across six cellular environments. This approach revealed strong cellular environment effects, but the method was not sensitive enough to detect small differences in the replication rate between the subtypes. By conducting pairwise competition experiments between the virus variants in six cellular environments, we could demonstrate significant differences in the replication rates of the subtypes and that LTR-determined viral fitness depends both on the host cell type and the activation state of the cell. In addition, we determined the degree of conservation of the transcription factor-binding sites (TFBS) in the different-subtype LTRs by analyzing sequences from the HIV sequence database. The sequence analyses revealed subtype-specific conservation of certain TFBS. The results indicate that one should consider the possibility of subtype-specific viral replication rates in vivo, which are strongly influenced by the host environment. We argue that the multidimensional host environment may have shaped the genetic structures of the subtype LTRs.

FIG. 1.

The HIV-1 LTR promoter in different subtypes. (A) Proviral genome of HIV-1 (not to scale). (B) A detailed picture of the subtype-specific TFBS in the U3 domain of the LTR promoter. Molecular clones were constructed through exchange of the U3 and R regions (nucleotides −147 to +63) of the subtype B molecular clone LAI with eight other subtype-specific promoter sequences. The U3 sequence changes do not affect the upstream Nef open reading frame. Because the variant sequences were introduced into the 3′ LTR of subtype B, the R element encoding TAR is inherited from the 5′ LTR. The asterisks beneath each TFBS indicate the predicted fit obtained by MatInspector, as follows: ***, good; **, average; *, poor. —, no prediction.

FIG. 2.

The subtype LTR directs differential viral replication. Virus replication was monitored by measuring CA-p24 production. (A) Replication curves of the nine virus variants in three different SupT1 cellular environments. (B) Replication in three different MT2 cellular environments.

FIG. 3.

Virus competition. The percentage of each virus in the competition was determined at five time points. SupT1 cells were infected with equal amounts of subtypes A and B (▵) or of subtypes A and E (▴). Only the change in the percentage of subtype A is shown.

FIG. 4.

Change in viral fitness as determined by the environment. Shown are the reaction norms for each subtype, which indicate the change in relative fitness in a changing environment. The relative fitness values are depicted in Table 3. ▪, subtype A; ▴, subtype B; ×, subtype C1; ◊, subtype C2; □, subtype D; ▵, subtype E; •, subtype F; ○, subtype G; ⧫, subtype AG.

FIG. 5.

Conserved fitness and environmental responsiveness in different subtype C and E strains. The change in relative fitness was determined for two additional subtype E (E-Fin24 and E-Fin4) and two subtype C (C-Eth26 and C-Eth9) strains in SupT1 cells and SupT1 cells plus TNF-α. Relative fitness and the responsiveness to a changing environment are very similar for the genotypes belonging to the same subtype. ⧫, C2; ▪, C-Eth26; ▴, C-Eth9; ▵, E; ◊, E-Fin24; □, E-Fin4.