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Related post: initiation codon of HA. One mutant was of particular interest because it sustained an in-phase deletion. This in-phase mutant HA sustained a deletion of 33 bp or 11 amino acids, all within the signal peptide. Sequence analysis predicted that 5 amino acids of the 16 amino-acid signal sequence remained as a result of this deletion. The signal peptide cleavage site of Gly-Gln and the subsequent downstream sequences were not affected. Mutant HA lacking lacking 11 amino acids of signal sequence accumulated in the cytoplasm but was not inserted into the outer membrane. The mutant polypeptide synthesized during infection was not modified by glycosylation. Its failure to agglutinate red blood cells suggests that the mutant HA remained monomeric since the trimeric structure is required for functional activity. This functional defect of mutant HA may be due to the lack of carbohydrate components which are required for proper assembly (Sekikawa, Lai). Attempts to rescue cloned influenza virus DNA by allele replacement . Recently, cloned, full-length DNA derived from the single stranded RNA genome of poliovirus was shown to be infectious by Racaniello and Baltimore. This means that mutations introduced at strategic locations in full-length poliovirus cDNA can now be studied for their effect on viral structure, function and virulence. Similarly, our goal with influenza virus is to devise procedures that would permit conversion of influenza DNA to influenza virus genomic (negative strand) RNA and then transfer such RNA into an influenza virus. In this manner, stable site-specific mutations, such as deletions, induced in the cloned DNA, might be transferred back to the influenza virus. It might then be possible to develop stable mutants for experimental study and for use in immunoprophylaxis. To determine whether cloned influenza DNA could be converted back to viral RNA (vRNA) and packaged in the virion, influenza gene rescue experiments (so-called allele replacement) were attempted employing the following protocol. Recombinant SV40-HA and SV40-NA DNA were used because of the ease of identification of the two surface antigens. Also specific antiserum which effectively neutralized virus bearing HA or NA of the coinfecting virus could be used to facilitate detection of reassortant viruses that had undergone allele replacement. Permissive African green monkey kidney cells were infected sequentially with a SV40-HA (H3) or SV40-NA (N2) recombinant followed by influenza A/WSN/1933 (HlNl) which bore surface antigens of another subtype. Infected cell lysates were passaged once and incubated with WSN antiserum to neutralize progeny virus bearing HI or Nl and thus favor the detection of reassortant virus Grisactin 500 that had undergone allele replacement. Heterogeneous WSN antiserum (anti-HlNl) was used in the attempt at allele replacement of the HI gene, while monoclonal antibody capable of neutralizing virus bearing Nl was used in the attempt at replacement of the Nl gene. This protocol should allow detection of reassortant virus that had acquired an RNA segment derived from a HA or NA DNA insert resulting in replacement of the corresponding WSN gene. When SV40-HA or SV40-NA was tested in this manner, rescued virus could not be identified. Using SV40-NA recombinants which produced either a positive strand 22-15 or a negative strand RNA transcript, we were unsuccessful in detecting rescued virus. Positive full-length influenza RNA sequences were produced from the former orientation, but they were flanked at both ends by S\/40 sequences (5' late SV40 transcription initiation sequences and 3' polyadenylation signals). It is possible that the viral replicase provided by the coinfecting influenza virus did not recognize the terminal influenza sequences because of these flanking SV40 sequences and was thus unable to start influenza vRNA synthesis. Similarly, failure to rescue negative RNA transcripts produced during infection by SV40-HA or SV40-NA with an influenza insert in the opposite orientation, suggests that influenza transcripts presumably containing flanking SV40 sequences were not encapsidated, replicated and packaged in virions. Precise terminal sequences may be a prerequisite for transcription, replication, and encapsidation of full-length influenza RNA. If this is the case, the 5' and 3' flanking SV40 sequences contained in RNA transcripts from the recombinant influenza DNA may have prevented rescue. In order to provide specific terminal sequences in the RNA transcripts that are recognized by the influenza replicase complex, it may be necessary to remove the flanking S\/40 sequences (Lai, Markoff, Sveda, Chanock). Persistent expression of influenza virus nucleoprotein (NP) in eukaryotic cells . Selective complementation of defective influenza A virus mutants and ultimately, "rescue" of cloned mutant influenza DNA into influenza virions may require the expression of cloned genes in persistently infected, stably transformed cells. For example, one obstacle to achieving complementation and rescue during lytic infection by our established SV40 vectors is interference of co-infecting influenza virus by replicating SV40 . Bovine papilloma virus (BPV) is a large DNA virus that replicates autonomously and extrachromosomally during persistent infection of animal cells. Recently a collaborative effort was initiated with Drs. Peter Howley and Ming-fan Law (NCI) in an effort to exploit their BPV vector for the expression of cloned influenza viral genes. A BPV recombinant that incorporated the influenza nucleoprotein (NP) gene was successfully constructed. The BPV-NP recombinant DNA was used to transform mouse C127 cells. Infected cells were analyzed for the production of the NP protein by (1) polyacrylamide gel electrophoresis in order to estimate molecular size, (2) indirect immunofluorescence in order tQ2establish Buy Grisactin the location of NP protein in infected cells and (3) labelling with P orthophosphate in order to detect post-translational modification of NP protein. It was shown that (1) protein from BPV-NP transformed cells was immunoprecipitated specifically by NP monoclonal antibodies; (2) a labelled protein of 56K daltons equivalent in size to the NP produced during influenza virus infection was produced in BPV-NP infected cells, while such a protein was not formed in BPV transformed cells; (3) the NP protein localized in the nucleus and cytoplasm of BPV-NP transformed cell; (4) the NP protein was also phosphorylated in the BPV-NP transformed cell. The mouse cell line (C127) used in the initial studies is not permissive for influenza virus replication and hence can not be employed for gene rescue ("allele replacement"). For this reason, other host cell systems were examined to determine if they could support both influenza virus replication and transfection by BPV. Simian CV-1 cells show some promise in this regard. CV-1 cells were co-transfected with the BPV-NP recombinant and a neomycin resistance
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