Comparison of SIV and HIV-1 genomic RNA structures reveals impact of sequence evolution on conserved and non-conserved structural motifs

RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1NL4-3. One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1NL4-3 also occur at the 5′ polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve.
Author Summary We have taken advantage of the rapid evolution of primate lentiviruses to assess the conservation of secondary structure in the viral RNA genome. We determined the structure of the SIVmac239 RNA genome to allow a detailed comparison with the previously determined structure of the HIV-1NL4-3 genome. In comparing the two structures, we find very few conserved base pairs with the same pairing partners, indicating that RNA structure is evolving even faster than the sequence. This suggests that most of the genome is in a metastable state that refolds during sequence evolution. Specific areas of structure that are required for function are maintained by the clustering of guanosines in the otherwise adenosine-rich genome, although the precise organization of the structure evolves. The strong effect of selection on maintainence of protein recognition sites can be seen in the conservation of pairing partners within the Rev binding sites in the RRE RNA. We propose that the more stable elements of RNA structure that are needed for function are susceptible to mutation during viral DNA synthesis. This causes the structures to evolve rapidly, yet still within the constricts of selective pressure, allowing maintenance of function.