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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
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
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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