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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.

SUBMITTER: Takata MA

PROVIDER: S-EPMC5805364 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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

Takata Matthew A MA Soll Steven J SJ Emery Ann A Blanco-Melo Daniel D Swanstrom Ronald R Bieniasz Paul D PD

PLoS pathogens 20180129 1

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 ...[more]

PMID: 29377940

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Project description:Retroviruses are well known for their ability to incorporate envelope (Env) proteins from other retroviral strains and genera, and even from other virus families. This characteristic has been widely exploited for the generation of replication-defective retroviral vectors, including those derived from murine leukemia virus (MLV), bearing heterologous Env proteins. We investigated the possibility of "genetically pseudotyping" replication-competent MLV by replacing the native env gene in a full-length viral genome with that of another gammaretrovirus. Earlier, we developed replication-competent versions of MLV that stably transmit and express transgenes inserted into the 3' untranslated region of the viral genome. In one such tagged MLV expressing green fluorescent protein, we replaced the native env sequence with that of gibbon ape leukemia virus (GALV). Although the GALV Env protein is commonly used to make high-titer pseudotypes of MLV vectors, we found that the env replacement greatly attenuated viral replication. However, extended cultivation of cells exposed to the chimeric virus resulted in selection of mutants exhibiting rapid replication kinetics and different variants arose in different infections. Two of these variants had acquired mutations at or adjacent to the splice acceptor site, and three others had acquired dual mutations within the long terminal repeat. Analysis of the levels of unspliced and spliced viral RNA produced by the parental and adapted viruses showed that the mutations gained by each of these variants functioned to reverse an imbalance in splicing caused by the env gene substitution. Our results reveal the presence of previously unknown cis-acting sequences in MLV that modulate splicing of the viral transcript and demonstrate that tagging of the retroviral genome with an easily assayed transgene can be combined with in vitro evolution as an approach to efficiently generating and screening for replicating mutants of replication-impaired recombinant viruses.

Project description:Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5'-SLA and 5'-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

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

Publications

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

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