Global synonymous mutagenesis identifies cis-acting RNA elements that regulate HIV-1 splicing and replication

Abstract

The ~9.5 kilobase HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication, including transcription, splicing, nuclear export, translation, packaging and reverse transcription. Nonetheless, chemical probing and other approaches suggest that the HIV-1 genome may contain many more RNA secondary structures of unknown importance and function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we undertook a global silent mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice site(s). Overall, our data indicate complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication.

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

“…The introduced CpGs do not directly enhance the strength of this splice site because they are upstream of the splice donor and do not affect the sequence itself. A previous report has identified that introducing synonymous mutations into the 5= end of gag promotes splicing at this cryptic donor, but the role of CpGs in this was not characterized (50). We found that CpGs introduced into this region have multiple effects on viral replication, including decreases in genomic-RNA stability, Gag expression, virion production, and infectivity per genome (15).…”

“…CpG dinucleotides introduced into gag can inhibit HIV-1 gene expression by modulating pre-mRNA splicing. The HIV-1 genomic RNA undergoes extensive alternative splicing to mediate expression of all of the viral genes, and synonymous mutations in gag have previously been shown to disrupt HIV-1 splicing (36,50). Since the CpGs in the 5= region of gag reduced Env expression in all sequence contexts tested ( Fig.…”

HIV-1 Protease Evolvability Is Affected by Synonymous Nucleotide Recoding

“…The HIV-1 genome exhibits a particularly striking bias toward enrichment of A-rich codons, which may be a selectable trait (36) and may affect innate immune recognition (11,29). Similarly, synonymous codon usage can temporally regulate the expression of structural gene products of SIV (31) and can regulate HIV-1 splicing and replication (12).…”

HIV-1 Protease Evolvability is Affected by Synonymous Nucleotide Recoding

“…Although they do not influence the resulting protein sequence, synonymous mutations can still substantially affect cellular processes (1, 2). Notably, synonymous virus genome recoding can impact viral replication capacity and fitness (3), reportedly leading to attenuation of multiple RNA and DNA viruses, including poliovirus (4-7), influenza virus (8, 9), HIV-1 (10-12), SIV (13), Chikungunya virus (14), human respiratory syncytial virus (15-17), porcine reproductive and respiratory syndrome virus (18), echovirus 7 (19, 20), tick-borne encephalitis virus (21), vesicular stomatitis virus, dengue virus (22), adeno-associated virus (23), and papillomavirus (24).…”

“…The HIV-1 genome exhibits a particularly striking bias towards enrichment of A-rich codons, which may be a selectable trait (36) and affect innate immune recognition (11, 29). Similarly, synonymous codon usage can temporally regulate expressions of structural gene products of the simian immunodeficiency virus (SIV) (31) and regulate HIV-1 splicing and replication (12).…”