Transcription
Eight expression plasmids (pHW2000) corresponding to the viral
genomic segments were transfected to express all influenza genes.
The first eight genetic constructs above are for the H1N1 fraction
and the later 8 are for the H3N2 fractions of the vaccine. The
plasmid contains bidirectional sets of expression cassettes:
Cytomegalovirus promoter - Bovine growth hormone terminator (sense)
and Human polymerase I promoter - Mus musculus polymerase
I terminator (antisense). The combination of transcription in both
directions and interaction with host cellular machinery results in
the production of infectious virions.
For protein expression of the influenza genes, transcription by RNA
polymerase II is directed from the Cytomegalovirus promoter and
terminates at the bovine hormone terminator. For segments VII and
VII (matrix protein 2 and nuclear export protein, respectively),
the transcripts will be spliced to join the initial coding
sequences to the later sequences and form a mature mRNA. The
transcripts receive 5' caps and 3' poly-adenylated tails. Thus, the
transcripts are translated into proteins. To produce viral RNA to
function as genomic material, transcription must produce negative
sense (antisense) cRNA. Thus, transcription is directed from the
human polymerase I promoter and terminates at the Mus
musculus polymerase I terminator. These transcripts have a 5'
triphosphate group and no polyadenylated tail. Thus, they are not
translated into proteins.
Sequence source
H1N1 modified virion (Lot no. 241-192) - first eight genetic
constructs above
Influenza strain A/swine/Minnesota/37866/1999 (H1N1): HA and NA
proteins
Influenza strain A/Swine/Texas/4199-2/98 (H3N2): PB1, PB2, PA, NP,
M1, M2, NS1 and NEP
H3N2 modified virion (Lot no. 241-196) - later eight genetic
constructs above
Influenza strain A/Swine/Texas/4199-2/98 (H3N2): PB1, PB2, PA, HA,
NP, NA, M1, M2, NS1 and NEP
Note:
For both modified virions, the nonstructural protein 1 (NS1) has
been carboxyl-truncated and codes for amino acids 1 to 126 of the
native protein. The truncation is related to the attenuation
(reduction in virulence) of the seed virus.
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