Fase 1 – Aplicación de la Ingeniería del Conocimiento 81

Influenza virus replication is dependent on an active host cell nucleus both for viral transcription and for the processing of the viral mRNAs. The major host cell nuclear-dependent function involved in influenza virus replication is the synthesis of host cell mRNA which serves as a primer in the transcription of virus specific mRNA (Bouloy et al ., 1978; Krug et al., 1979). The initial transcription of viral RNA in the cell to form mRNAs [poly A(+)cRNA] is known as primary transcription. This system can be readily duplicated i ri vitro using disrupted virions.

Secondary transcription is dependent on protein synthesis and results in progeny RNA molecules. Here the virus copies its vRNA into full length cRNA transcripts [poly A(-)cRNA] which act as templates for an RNA replicase to produce full-length vRNA for transcription into progeny virus. The synthesis of the three different classes of viral RNAs (mRNA, poly A(-)cRNA and vRNA), and the relative abundance of specific RNAs and vRNAs are controlled throughout infection.

3.4.1. Early intracellular events: mRNA formation.

The first phase of influenza replication - primary transcription results in the synthesis of all eight mRNAs

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complementary "to each segment of RNA in approximately equimolar amounts. In less than 20 minutes after infection mRNAs can be detected (Hay et al., 1977; Barrett et al., 1979; Mark et al., 1979) and their rate of synthesis reaches a peak after 2 hours. Primary transcription is independent of protein synthesis and takes place in the nucleus (Herz et al., 1981). Subsequently

transcripts are transported to their site of function in the cytoplasm.

In order for transcription to take place cellular mRNAs are

required. Virion RNA transcription in vitro, catalyzed by the virion-associated transcriptase, is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG (McGeoch and Kitron, 1975; Plotch and Krug, 1977; 1978). in vitro , eukaryotic mRNAs and other RNAs containing a fully methylated cap

structure (Gm7 GpppXm ) stimulated the transcription of influenza viral RNA (Plotch et al., 1981). 5 terminal RNA fragments containing the cap and the ensuing 10-14 nucleotides are cleaved

from the host mRNA (e.g. beta-globulin mRNA) and are used as primers to initiate viral RNA transcription. The virion contains a cap-dependent endonuclease that cleaves capped RNAs preferentially at a purine residue that is 10-13 nucleotides from the 5 terminus. The mRNAs therefore have a 5' terminal type I cap structure (Krug et al., 1976). The priming of influenza viral RNA transcription by capped host RNAs also occurs in the infected cell (Krug et al., 1979). Host cell mRNAs and/or the precursors serve as primers for viral RNA transcription in the infected cell

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and donate their cap and 10-15 nucleotides, one of which is m6 A, to resulting viral mRNA molecules.

The 5' ends of influenza viral RNAs have to be donated from newly synthesized capped host RNA species, as pre-existing RNAs in the nucleus remaining after the addition of alpha amanitin do not prime (alpha amanitin inhibits DNA-dependent RNA polymerase II). Two of the influenza mRNA species are further modified by splicing (presumably by host cell enzymes) which allows overlapping regions of the genome to direct synthesis of additional polypeptides. In this manner NS2 is derived from segment 8 and M2 is derived from segment 7 (Lamb and Lai, 1980, Lamb et a l ., 1981).

The process of cleavage of the capped primer RNA, initiation, elongation and termination/polyadenylation of the transcript are all carried out by the proteins of the viral core (Plotch et a l ., 1981) i.e. NP, PB1, PB2 and PA (sections 1.3; 2.1; 2.3). NP is considered to have a structural role, PB2 is the cap recognition protein (Ulmanen et a l ., 1981; Braam-Markson et al., 1985). PB1 probably catalyzes the initiation of transcription (Ulmanen et a l . , 1981) and then acts as the transcriptase enzyme, adding nucleotides. It has not been determined which viral protein(s) act as the endonuclease (i.e. cleaving the capped RNA) but PB2 (in recognizing the 5 cap) and PB1 (in perhaps cleaving the phosphate bond) may constitute the

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•transcriptase complex catalyzing mRNA synthesis that moves down

the growing viral mRNA chain (Braam et al, 1983), a specific function for the PA protein has not yet been identified.

3.4.2. Template poly A-cRNA synthesis.

A second population of RNA transcripts [poly A(-)cRNA] are

detected after the mRNAs appear in the cell and are synthesized whilst mRNAs are being produced. Synthesis of poly A(-)cRNA reaches a maximum rate 30-90 minutes post infection, before the peak of mRNA production. Poly A(-)cRNA molecules are complementary to the vRNA segments found in infected cells. They are complete transcripts of the vRNA segments, lack host derived primer sequences, are non-polyadenylated and probably act as templates for vRNA synthesis. The full length transcripts are probably also synthesized in the nucleus (Herz et al., 1981). As the synthesis of these transcripts is inhibited by cycloheximide

it is suggested that synthesis is dependent upon a virus-specified protein(s) which perhaps modify the transcriptase

complex so that transcription is initiated without a primer.

3.4.3. vRNA Synthesis.

Naturally enough the synthesis of vRNAs follows poly A(-)cRNA production, although the precise timing of events is uncertain. The actual mechanisms by which vRNAs are replicated is not understood but synthesis is dependent on viral protein (Hay et al., 1977; Barrett et al., 1979; 1979; Mark et al., Mark et al., 1979). The

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location of the site of synthesis of vRNA has also not been delineated. Segments can be detected both in the cytoplasm and the nucleus. vRNA segments are produced in non-equivalent amounts.

There is a correspondance between vRNA and mRNA synthesis suggests that the two events may be closely coupled (Smith and Hay, 1982). The time at which cRNA is synthesized suggests that the the template for synthesis of mRNA in secondary transcription is newly synthesized vRNA and that cRNA is synthesized only from input genome RNA (Wolstenholme et al., 1980).

The characteristics of influenza virus replication can differ depending on the host cell (Smith and Hay, 1982). There are also differences between virus strains (Lamb and Choppin, 1977; Hay et al., 1977). Sub-genomic RNAs which interfere with subsequent virus multiplication are sometimes synthesized during replication. These are defective-interfering (DI) RNAs (Nayak and Chambers, 1985). The generation of DI RNAs is favoured by high multiplicity and the host cell (Choppin and Pons, 1970; Janda et al., 1979) which may reflect a function provided by the host cell

for replication.