The 5′-cap structures of eukaryotic m RNAs are important for RNA stability, pre-m RNA splicing,m RNA export, and protein translation. Many viruses have evolved mechanisms for generating their own cap structures with ...The 5′-cap structures of eukaryotic m RNAs are important for RNA stability, pre-m RNA splicing,m RNA export, and protein translation. Many viruses have evolved mechanisms for generating their own cap structures with methylation at the N7 position of the capped guanine and the ribose 2′-Oposition of the first nucleotide, which help viral RNAs escape recognition by the host innate immune system. The RNA genomes of coronavirus were identified to have 5′-caps in the early1980 s. However, for decades the RNA capping mechanisms of coronaviruses remained unknown.Since 2003, the outbreak of severe acute respiratory syndrome coronavirus has drawn increased attention and stimulated numerous studies on the molecular virology of coronaviruses. Here, we review the current understanding of the mechanisms adopted by coronaviruses to produce the 5′-cap structure and methylation modification of viral genomic RNAs.展开更多
Many flaviviruses are significant human pathogens.The plus-strand RNA genome of a flavivirus contains a 5′terminal cap 1 structure(m7GpppAmG).The flavivirus encodes one methyltransferase(MTase),located at the Ntermin...Many flaviviruses are significant human pathogens.The plus-strand RNA genome of a flavivirus contains a 5′terminal cap 1 structure(m7GpppAmG).The flavivirus encodes one methyltransferase(MTase),located at the Nterminal portion of the NS5 RNA-dependent RNA polymerase(RdRp).Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development.The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation.Representative flavivirus MTases,from dengue,yellow fever,and West Nile virus(WNV),sequentially generate GpppA!m7GpppA!m7GpppAm.Despite the existence of two distinct methylation activities,the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine(SAM),the methyl donor.This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions.Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide.Mutant enzymes with different methylation defects can trans complement one another in vitro,demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro.In the context of the infectious virus,defects in both methylations,or a defect in the N7 methylation alone,are lethal to WNV.However,viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-typeWNV challenge.The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and,thus,methyltransferase represents a novel and promising target for flavivirus therapy.展开更多
Background The capsule associated protein 10 gene （caplO） is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus （C.） neoformans. In this st...Background The capsule associated protein 10 gene （caplO） is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus （C.） neoformans. In this study, we aimed to construct an short hairpin RNA （shRNA） expression vector targeting C. neoformans caplO gene expression and confirm its biologic relevance. Methods A pair of oligonucleotides targeting the cap10 cDNA sequence was designed and synthesized. It was cloned into the plasmid psilencer4.1-CMV neo to construct an eukaryotic shRNA expression vector. The vector was transfected into C. neoformans cells using the LiAc method. The expression of cap10 was assessed by real-time fluorescence quantitative PCR. Groups of C. neoformans cells were incubated with murine macrophage-like J774A.1 cells, and the phagocytic indexes and ratios were determined by the microscopic observation method. Results The expression of cap10 in C. neoformans cells transfected with ps4.1 nee-cap10 （（175 535.00±47 004.00） copies/μl） was lower than that of cells transfected with the empty vector （（512 698.89±32 318.02） copies/μl） and mock transfected cells （（562 931.66±65 928.41） copies/μl）. The average phagocytic ratio and phagocytic index of J774A.1 cells following incubation with C. neoformans were higher for cells transfected with ps4.1 neo-capl0 （0.21±0.02, （19.06±1.66）%） than for the control experimental group （0.08±0.02, （6.57±1.23）%） and the blank experimental group （（0.07±0.01）, （5.89±1.07）%） （P 〈0.05）. Conclusions The cap10 shRNA vector was successfully prepared and transfected into C. neoformans cells. The effect of RNA interference on the expression of the C. neoformans caplO gene is effective, and it can induce phagocytosis of C. neoformans.展开更多
基金supported by the China "973" Basic Research Program (2013CB911101)China NSFC grants (81130083 and 81271817)
文摘The 5′-cap structures of eukaryotic m RNAs are important for RNA stability, pre-m RNA splicing,m RNA export, and protein translation. Many viruses have evolved mechanisms for generating their own cap structures with methylation at the N7 position of the capped guanine and the ribose 2′-Oposition of the first nucleotide, which help viral RNAs escape recognition by the host innate immune system. The RNA genomes of coronavirus were identified to have 5′-caps in the early1980 s. However, for decades the RNA capping mechanisms of coronaviruses remained unknown.Since 2003, the outbreak of severe acute respiratory syndrome coronavirus has drawn increased attention and stimulated numerous studies on the molecular virology of coronaviruses. Here, we review the current understanding of the mechanisms adopted by coronaviruses to produce the 5′-cap structure and methylation modification of viral genomic RNAs.
基金This research was partially supported by grants from the National Institute of Health(NIH)(No.AI07079201A1)to H.L。
文摘Many flaviviruses are significant human pathogens.The plus-strand RNA genome of a flavivirus contains a 5′terminal cap 1 structure(m7GpppAmG).The flavivirus encodes one methyltransferase(MTase),located at the Nterminal portion of the NS5 RNA-dependent RNA polymerase(RdRp).Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development.The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation.Representative flavivirus MTases,from dengue,yellow fever,and West Nile virus(WNV),sequentially generate GpppA!m7GpppA!m7GpppAm.Despite the existence of two distinct methylation activities,the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine(SAM),the methyl donor.This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions.Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide.Mutant enzymes with different methylation defects can trans complement one another in vitro,demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro.In the context of the infectious virus,defects in both methylations,or a defect in the N7 methylation alone,are lethal to WNV.However,viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-typeWNV challenge.The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and,thus,methyltransferase represents a novel and promising target for flavivirus therapy.
文摘Background The capsule associated protein 10 gene （caplO） is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus （C.） neoformans. In this study, we aimed to construct an short hairpin RNA （shRNA） expression vector targeting C. neoformans caplO gene expression and confirm its biologic relevance. Methods A pair of oligonucleotides targeting the cap10 cDNA sequence was designed and synthesized. It was cloned into the plasmid psilencer4.1-CMV neo to construct an eukaryotic shRNA expression vector. The vector was transfected into C. neoformans cells using the LiAc method. The expression of cap10 was assessed by real-time fluorescence quantitative PCR. Groups of C. neoformans cells were incubated with murine macrophage-like J774A.1 cells, and the phagocytic indexes and ratios were determined by the microscopic observation method. Results The expression of cap10 in C. neoformans cells transfected with ps4.1 nee-cap10 （（175 535.00±47 004.00） copies/μl） was lower than that of cells transfected with the empty vector （（512 698.89±32 318.02） copies/μl） and mock transfected cells （（562 931.66±65 928.41） copies/μl）. The average phagocytic ratio and phagocytic index of J774A.1 cells following incubation with C. neoformans were higher for cells transfected with ps4.1 neo-capl0 （0.21±0.02, （19.06±1.66）%） than for the control experimental group （0.08±0.02, （6.57±1.23）%） and the blank experimental group （（0.07±0.01）, （5.89±1.07）%） （P 〈0.05）. Conclusions The cap10 shRNA vector was successfully prepared and transfected into C. neoformans cells. The effect of RNA interference on the expression of the C. neoformans caplO gene is effective, and it can induce phagocytosis of C. neoformans.
