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The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed -1 ribosomal frameshifting.

ABSTRACT: Several important viruses including the human immunodeficiency virus type 1 (HIV-1) and the SARS-associated Coronavirus (SARS-CoV) employ programmed -1 ribosomal frameshifting (PRF) for their protein expression. Here, a kinetic framework is developed to describe -1 PRF. The model reveals three kinetic pathways to -1 PRF that yield two possible frameshift products: those incorporating zero frame encoded A-site tRNAs in the recoding site, and products incorporating -1 frame encoded A-site tRNAs. Using known kinetic rate constants, the individual contributions of different steps of the translation elongation cycle to -1 PRF and the ratio between two types of frameshift products were evaluated. A dual fluorescence reporter was employed in Escherichia coli to empirically test the model. Additionally, the study applied a novel mass spectrometry approach to quantify the ratios of the two frameshift products. A more detailed understanding of the mechanisms underlying -1 PRF may provide insight into developing antiviral therapeutics.

SUBMITTER: Liao PY

PROVIDER: S-EPMC3017607 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed -1 ribosomal frameshifting.

Liao Pei-Yu PY Choi Yong Seok YS Dinman Jonathan D JD Lee Kelvin H KH

Nucleic acids research 20100907 1

Several important viruses including the human immunodeficiency virus type 1 (HIV-1) and the SARS-associated Coronavirus (SARS-CoV) employ programmed -1 ribosomal frameshifting (PRF) for their protein expression. Here, a kinetic framework is developed to describe -1 PRF. The model reveals three kinetic pathways to -1 PRF that yield two possible frameshift products: those incorporating zero frame encoded A-site tRNAs in the recoding site, and products incorporating -1 frame encoded A-site tRNAs. U ...[more]

PMID: 20823091

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Project description:The -2/-1 programmed ribosomal frameshifting (-2/-1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This -2/-1 PRF mechanism is transactivated by a viral protein, nsp1β, and cellular poly(rC) binding proteins (PCBPs). Critical elements for -2/-1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate -2 PRF to generate nsp2TF. The nsp1β of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both -2 and -1 PRF, and the universally conserved Tyr111 and Arg114 in nsp1β are essential for this activity. In vitro translation experiments demonstrated the involvement of PCBPs in simarterivirus -2/-1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1β, slippery sequence, and C-rich motif in -2/-1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of -2/-1 PRF are highly conserved in all known arteriviruses except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, -2/-1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1βs of all non-EAV arteriviruses tested. Taken together, these data suggest that -2/-1 PRF is an evolutionarily conserved mechanism employed in non-EAV/-WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication.IMPORTANCE Simarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species, and some of them cause lethal hemorrhagic fever disease, this group of viruses has been undercharacterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be preemergent zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved ribosomal frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.

Dataset Information

The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed -1 ribosomal frameshifting.

Publications

The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed -1 ribosomal frameshifting.

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