Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes - PubMed (original) (raw)
Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes
Tokiko Watanabe et al. J Virol. 2003 Oct.
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.
Figures
FIG. 1.
Schematic diagram of mutant HA vRNAs and their efficiency of virion incorporation. All mutant HA RNAs are shown in the negative-sense orientation. Each mutant contains the GFP open reading frame (inserted in frame with the HA open reading frame) flanked by a stop codon, 33 nucleotides of the 3′ noncoding region, and 45 nucleotides of the 5′ noncoding region of HA vRNA. The lengths of the regions are not drawn to scale. Mutants were designated according to the number of nucleotides derived from the HA coding regions (orange bars). Horizontal broken lines indicate a deletion. The efficiency of incorporation of mutant HA vRNA into VLPs was determined by dividing the number of cells expressing GFP by that of cells expressing NP in the VLP-infected cells after the cells were fixed at 16 h postinfection. The levels of GFP expression were comparable among the constructs tested. The means of three experiments are shown.
FIG. 2.
The vRNA levels in 293T cells transfected with plasmids expressing mutant HA vRNAs. 293T cells were transfected with pPolIHA(0)GFP(0) or pPolIHA(9)GFP(80) and plasmids expressing PA, PB1, PB2, and NP. At 24 h posttransfection, vRNA present in transfected cells was extracted, and glyoxalated RNA was separated by electrophoresis on 1.0% agarose gel in 10 mM phosphate buffer. RNAs were blotted onto nylon membranes and hybridized with a digoxigenin-labeled probe complementary to the GFP sequence for overnight at 42°C. The RNA bands were detected by using DIG Nucleic Acid Detection Kit (Roche). Control RNA was extracted from mock-transfected 293T cells.
FIG. 3.
VSVG(HA)GFP(NA) virus-infected cells express VSVG and GFP. MDCK cells were infected with VSVG(HA)GFP(NA) virus or WSN virus and overlaid with 1.0% agarose. The infected cells were incubated for 48 h at 37°C, and representative plaques were photographed under normal light (A and B) and under fluorescent light (C and D). The cells were fixed and treated with 0.1% Triton X-100 in 3% formaldehyde solution. The viral proteins were detected by immunostaining with anti-WSN HA monoclonal antibody (E and F), anti-VSVG monoclonal antibody (G and H), or anti-WSN NP monoclonal antibody (I and J) as the primary antibody and biotinylated secondary antibody by using the Vectastain ABC kit.
FIG. 4.
Incorporation of the VSVG protein into VSVG(HA)GFP(NA) virus. Concentrated WSN and VSVG(HA)GFP(NA) viruses, and VSV were lysed in a sample buffer. Viral proteins were treated with 2-mercaptoethanol, separated by SDS-10% polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubated with anti-VSVG monoclonal antibody or anti-WSN HA monoclonal antibody.
FIG. 5.
Electron microscopy of VSVG(HA)GFP(NA) virus. VSVG(HA)GFP(NA) (A) and WSN (B) viruses were centrifuged through 20% sucrose, and the pelleted materials were then negatively stained with 2% PTA. (C) Pelleted VSVG(HA)GFP(NA) virus was also immunolabeled with anti-VSVG monoclonal antibody conjugated to 15-nm gold particles. Bar, 100 nm.
FIG. 6.
Growth curves of VSVG(HA)GFP(NA) virus in BHK, CHO, and MDCK cells. BHK (A), MDCK (B), and CHO (C) cells were infected with virus at a multiplicity of infection of 0.001. At the indicated times after infection, the virus titer in the supernatant was determined by using MDCK cells. The values are means of duplicate experiments.
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