scholarly journals Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions

2006 ◽  
Vol 80 (5) ◽  
pp. 2318-2325 ◽  
Author(s):  
Yukiko Muramoto ◽  
Ayato Takada ◽  
Ken Fujii ◽  
Takeshi Noda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
...  
Keyword(s):  
Influenza A ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Viral Rna ◽  
Green Fluorescent Protein Gene ◽  
Influenza A Viruses ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Green Fluorescent ◽  
Efficient Viral Replication

ABSTRACT The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.

scholarly journals Exploitation of Nucleic Acid Packaging Signals To Generate a Novel Influenza Virus-Based Vector Stably Expressing Two Foreign Genes

2003 ◽  
Vol 77 (19) ◽  
pp. 10575-10583 ◽  
Author(s):  
Tokiko Watanabe ◽  
Shinji Watanabe ◽  
Takeshi Noda ◽  
Yutaka Fujii ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Influenza Viruses ◽  
Coding Region ◽  
Influenza A Viruses ◽  
Virion Incorporation ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Genomic Regions

ABSTRACT At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007, 2003). To determine the signals in the hemagglutinin (HA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3′ end of the coding region and 80 nucleotides at the 5′ end are sufficient for efficient virion incorporation of the HA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose HA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of HA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.

scholarly journals Specific Residues of the Influenza A Virus Hemagglutinin Viral RNA Are Important for Efficient Packaging into Budding Virions

2007 ◽  
Vol 81 (18) ◽  
pp. 9727-9736 ◽  
Author(s):  
Glenn A. Marsh ◽  
Raheleh Hatami ◽  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Viral Rna ◽  
Wild Type ◽  
Reading Frame ◽  
Synonymous Mutations ◽  
Green Fluorescent ◽  
Ha Protein ◽  
Virus Replication Cycle

ABSTRACT A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3′ and 80 5′ nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.

scholarly journals Nucleotide Sequence Requirements at the 5′ End of the Influenza A Virus M RNA Segment for Efficient Virus Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3384-3388 ◽  
Author(s):  
Makoto Ozawa ◽  
Junko Maeda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
Shinji Watanabe ◽  
Hideo Goto ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Viral Rna ◽  
Coding Region ◽  
Noncoding Regions ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Sequence Requirements

ABSTRACT The mechanism by which the influenza A virus genome is packaged into virions is not fully understood. The coding and noncoding regions necessary for packaging of the viral RNA segments, except for the M segment, have been identified. Here, we delineate the M segment regions by incorporating a reporter viral RNA into virions and by generating viruses possessing mutations in the regions. We found that, like the other segments, the M segment coding regions are essential for virion incorporation and that the nucleotide length rather than the nucleotide sequence of the 5′ end of the coding region is important.

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

2014 ◽  
Vol 16 (6) ◽  
pp. 674-683 ◽  
Author(s):  
Chao Qiu ◽  
Bin Cheng ◽  
Yunsheng Zhang ◽  
Rong Huang ◽  
Lanjie Liao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

scholarly journals Response of immunocompetent and immunosuppressed Spodoptera littoralis larvae to baculovirus infection

2006 ◽  
Vol 87 (8) ◽  
pp. 2217-2225 ◽  
Author(s):  
Hadassah Rivkin ◽  
Jeremy A. Kroemer ◽  
Alexander Bronshtein ◽  
Eduard Belausov ◽  
Bruce A. Webb ◽  
...  
Keyword(s):  
Immune System ◽  
Immune Responses ◽  
Spodoptera Littoralis ◽  
Fluorescent Protein ◽  
Oral Route ◽  
Green Fluorescent Protein Gene ◽  
Successful Infection ◽  
Baculovirus Infection ◽  
Budded Virus ◽  
Green Fluorescent

The Mediterranean lepidopteran pest Spodoptera littoralis is highly resistant to infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via the oral route, but highly sensitive to infection with budded virus (BV) via the intrahaemocoelic route. To study the fate of AcMNPV infection in S. littoralis, vHSGFP, an AcMNPV recombinant that expresses the reporter green fluorescent protein gene under the control of the Drosophila heat-shock promoter, and high-resolution fluorescence microscopy were utilized. S. littoralis fourth-instar larvae infected orally with vHSGFP showed melanization and encapsulation of virus-infected tracheoblast cells serving the midgut columnar cells. At 72 h post-infection, the viral foci were removed during the moult clearing the infection. Thus, oral infection was restricted by immune responses to the midgut and midgut-associated tracheal cells. By contrast, injection of BV into the haemocoel resulted in successful infection of tracheoblasts, followed by spread of the virus through the tracheal epidermis to other tissues. However, in contrast to fully permissive infections where tracheoblasts and haemocytes are equally susceptible to infection, a severe limitation to vHSGFP infection of haemocytes was observed. To investigate the resistance of S. littoralis haemocytes to BV infection with AcMNPV, the larval immune system was suppressed with the Chelonus inanitus polydnavirus or a putatively immunosuppressive polydnavirus gene, P-vank-1. Both treatments increased the susceptibility of S. littoralis larvae to AcMNPV. It is concluded that the resistance of S. littoralis to AcMNPV infection involves both humoral and cellular immune responses that act at the gut and haemocyte levels. The results also support the hypothesis that tracheolar cells mediate establishment of systemic baculovirus infections in lepidopteran larvae. The finding that polydnaviruses and their encoded genes synergize baculovirus infection also provides an approach to dissecting the responses of the lepidopteran immune system to viruses by using specific polydnavirus immunosuppressive genes.

Laser-induced gene expression in specific cells of transgenic zebrafish

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1953-1960 ◽  
Author(s):  
M.C. Halloran ◽  
M. Sato-Maeda ◽  
J.T. Warren ◽  
F. Su ◽  
Z. Lele ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Transgenic Zebrafish ◽  
Hsp70 Promoter ◽  
Expression Of Genes ◽  
Transgenic Lines ◽  
Transgenic Embryos ◽  
Green Fluorescent

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

2009 ◽  
Vol 72 (7) ◽  
pp. 1513-1520 ◽  
Author(s):  
MANAN SHARMA ◽  
DAVID T. INGRAM ◽  
JITENDRA R. PATEL ◽  
PATRICIA D. MILLNER ◽  
XIAOLIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Green Fluorescent Protein Gene ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Novel Approach ◽  
Efficient Uptake ◽  
Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

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