The Experts below are selected from a list of 93 Experts worldwide ranked by ideXlab platform

Teryl K. Frey - One of the best experts on this subject based on the ideXlab platform.

  • Development of a Rubella Virus Vaccine Expression Vector: Use of a Picornavirus Internal Ribosome Entry Site Increases Stability of Expression
    Journal of virology, 2000
    Co-Authors: Konstantin V. Pugachev, Wen-pin Tzeng, Teryl K. Frey
    Abstract:

    Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other Togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.

Konstantin V. Pugachev - One of the best experts on this subject based on the ideXlab platform.

  • Development of a Rubella Virus Vaccine Expression Vector: Use of a Picornavirus Internal Ribosome Entry Site Increases Stability of Expression
    Journal of virology, 2000
    Co-Authors: Konstantin V. Pugachev, Wen-pin Tzeng, Teryl K. Frey
    Abstract:

    Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other Togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.

  • Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression
    2000
    Co-Authors: Konstantin V. Pugachev, Wen-pin Tzeng, K. Frey
    Abstract:

    Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Toga-viridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently devel-oped RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vac-cine vectors with other Togaviruses, the subgenomic promoter was duplicated to produce a recombinant con-struct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, ap-parently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, enceph-alomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construc-tion was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and unde

Hong Z Zhou - One of the best experts on this subject based on the ideXlab platform.

  • chaperone fusion proteins aid entropy driven maturation of class ii viral fusion proteins
    Trends in Microbiology, 2014
    Co-Authors: Hong Z Zhou
    Abstract:

    Class II viral fusion proteins are present on the envelope of flaviviruses and Togaviruses, viruses that often cause tropical and subtropical diseases. These proteins use a second membrane protein as a molecular chaperone to assist their folding and to ensure proper function during viral assembly, maturation, and infection. Recent progress in structural studies of dengue viruses has revealed how the chaperone pre-membrane (prM) protein guides viral maturation and how pH is sensed in both the maturation and infection processes. Drastic conformation changes and reorganization of these viral membrane proteins occur during the transition from their metastable to stable structural states in a unidirectional, entropy-driven process.

Nobuhiko Nakashima - One of the best experts on this subject based on the ideXlab platform.

  • translation initiation at the cuu codon is mediated by the internal ribosome entry site of an insect picorna like virus in vitro
    Journal of Virology, 1999
    Co-Authors: Jun Sasaki, Nobuhiko Nakashima
    Abstract:

    Many insect viruses that are morphologically and biophysically similar to mammalian picornaviruses have been reported (20), and they have been called insect picorna-like viruses. Recently, the complete nucleotide sequences of the genomes of several insect picorna-like viruses have been determined (7, 11, 19, 28, 37). Of these viruses, Drosophila C virus (DCV) (11), Rhopalosiphum padi virus (RhPV) (19) and Plautia stali intestine virus (PSIV) (28) were found to have a novel type of genome organization. The genomes of mammalian picornaviruses consist of positive-strand RNA containing a single large open reading frame (ORF) that codes for the capsid protein precursor in its 5′ part and the nonstructural protein precursor in its 3′ part (27). In contrast, the genomes of DCV, RhPV, and PSIV contain two ORFs that are separated by an intervening region (Fig. ​(Fig.1A).1A). The nonstructural protein precursor is encoded in the 5′-proximal ORF, and the capsid protein precursor is encoded in the 3′-proximal ORF. Previous studies of these three viruses have disclosed two unusual features concerning the translation of capsid proteins: the lack of an in-frame AUG initiation codon and the absence of subgenomic RNA (11, 19, 28). FIG. 1 Genome organization of PSIV. (A) Schematic diagram of the PSIV genome. ORFs are shown in open boxes. The numbers indicate nucleotide positions. The first nucleotide of the capsid protein gene represents the 5′-terminal nucleotide of the capsid-coding ... Several non-AUG initiation codons are used in the translation of viral and cellular mRNAs; however, their translation efficiency is generally lower than that of the AUG initiation codon (14). Most positive-strand RNA viruses have genome organizations that produce an excess of capsid proteins over nonstructural proteins. When capsid proteins are encoded in the 3′ part of the genome, as in caliciviruses and Togaviruses, the viruses produce subgenomic RNA to translate the capsid proteins (31). The excess production of capsid proteins is also observed in DCV in vivo (21), but the virus does not produce subgenomic RNA (11). These observations raise the question of how DCV, RhPV, and PSIV produce capsid proteins effectively. Previously, we showed that translation of the capsid protein of PSIV occurred independently of the nonstructural protein precursor and that the upstream region of the capsid protein gene was necessary for the translation (28). This observation suggested that the capsid protein of PSIV was translated by internal initiation. Internal initiation of translation was first characterized in mammalian picornavirus RNAs (10, 23). Picornavirus genomic RNAs lack the 5′ cap structure and have long 5′ untranslated regions (5′ UTR). The 5′ UTRs form multiple stem-loop structures, which are in contact with ribosomes. This region is called the internal ribosome entry site (IRES) and conducts cap-independent translation for protein synthesis from the genomic RNA (1, 2, 9, 30). Internal ribosome entry has also been reported for some viral and cellular RNAs such as hepatitis C virus (HCV) RNA (35, 38), cowpea mosaic virus RNA (33), and immunoglobulin heavy-chain binding protein mRNA (18). In this study, we confirmed that the capsid protein gene of PSIV was translated by internal ribosome entry in vitro by showing that translation of the gene occurs cap independently under dicistronic conditions. The 5′ and 3′ boundaries of the IRES were mapped, and the 3′ boundary was found to slightly overlap the capsid-coding region. We also identified the translation initiation codon of the capsid protein gene by using various site-directed mutants in vitro. The results indicated that translation of the capsid protein gene is initiated at the CUU codon that is located one codon upstream of the 5′ terminus of the capsid-coding region. CUU differs from AUG by two nucleotides, and such an AUG-unrelated initiation codon has not been reported to date. When the 5′ part of the IRES was deleted from a monocistronic RNA carrying the capsid protein gene, scanning ribosomes did not recognize the CUU codon. These data suggest that the PSIV IRES can effectively direct AUG-unrelated initiation.

Wen-pin Tzeng - One of the best experts on this subject based on the ideXlab platform.

  • Development of a Rubella Virus Vaccine Expression Vector: Use of a Picornavirus Internal Ribosome Entry Site Increases Stability of Expression
    Journal of virology, 2000
    Co-Authors: Konstantin V. Pugachev, Wen-pin Tzeng, Teryl K. Frey
    Abstract:

    Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other Togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.

  • Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression
    2000
    Co-Authors: Konstantin V. Pugachev, Wen-pin Tzeng, K. Frey
    Abstract:

    Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Toga-viridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently devel-oped RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vac-cine vectors with other Togaviruses, the subgenomic promoter was duplicated to produce a recombinant con-struct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, ap-parently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, enceph-alomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construc-tion was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and unde