Abstract

In RNA viruses, mutations occur fast and have large fitness effects. While this affords remarkable adaptability, it can also endanger viral survival due to the accumulation of deleterious mutations. How RNA viruses reconcile these two opposed facets of mutation is still unknown. Here we show that, in human immunodeficiency virus (HIV-1), spontaneous mutations are not randomly located along the viral genome. We find that the viral mutation rate experiences a threefold reduction in the region encoding the most external domains of the viral envelope, which are strongly targeted by neutralizing antibodies. This contrasts with the hypermutation mechanisms deployed by other, more slowly mutating pathogens such as DNA viruses and bacteria, in response to immune pressure. We show that downregulation of the mutation rate in HIV-1 is exerted by the template RNA through changes in sequence context and secondary structure, which control the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (A3)-mediated cytidine deamination and the fidelity of the viral reverse transcriptase.

Highlights

  • In RNA viruses, mutations occur fast and have large fitness effects

  • To test whether the HIV-1 mutation rate varies among regions of the envelope gene, we used a shuttle vector-based experimental system that allows for the accumulation of spontaneous mutations in cognate viral sequences in the absence of selection

  • Using both the shuttle vector and in vitro assays we found that the fidelity of the HIV-1 reverse transcriptase (RT) is dependent on RNA structure, and that differences in RNA structure in the region encoding the gp[120] external domains may further contribute to lowering the viral mutation rate

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Summary

Results and Discussion

We cloned the HIV-1 subtype A envelope (env) gene into a shuttle vector containing the minimal cis-acting elements required for transcription and encapsidation Following transfection of this construct into HEK 293T cells, pseudotyped viruses were generated by transient expression of transcomplementation plasmids encoding the HIV-1 proteins gag and pol, as well as the vesicular stomatitis virus envelope glycoprotein (VSV-G). This drop was driven by nucleotide substitutions, which occurred at 3.6-fold lower rate in the region encoding the gp[120] OA domains (15 in 1.0 kb) than in the rest of env (78 in 1.5 kb; Fisher test: Po0.001; Fig. 2c), whereas point insertions/deletions appeared to be more homogeneously distributed (four in the OA region versus seven in the rest of env; Fisher test: P40.5) This mutational cold spot was unexpected, because the gp[120] OA domains contain the most variable regions of the HIV-1 genome. G-A Other transitions Transversions Insertions Deletions Total mutations Bases sequenced Infection cycles Mutation rate (m/n/c)

C1 HR1 HR2
A B CA B C
Methods

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