After the onset of DNA synthesis, most late proteins are translated from mRNA's originating from the major late transcription unit (MLTU) which extends from the major late promoter (MLP) at map co-ordinate 16.8 (figure 1.6a) to a termination signal close to the right end of the genome. Although during the early phase of infection the MLP is active at a level comparable to other transcription units, transcription is subject to control at the level of transcriptional termination and does not extend beyond map co-ordinate 75. At late times post infection, the transcription termination block is alleviated (Larsson et a.l., 1992) and the primary transcript generated can be processed into a minimum of 20 mRNA's that are grouped into five families (LI to L5) according to their different polyadenylation sites (poly (A) sites).
An important event governing the transition from early to late gene expression is viral DNA replication. The intermediate gene product, protein IVa2, gains efficient access to viral templates during replication and contributes to the late phase dependant MLP activation. In
this respect, IVa2 is a temporal regulator, promoting late MLP activity and the generation of late (LI to L5) mRNA transcripts (Tribouley et al., 1994; Lutz and Kedinger, 1995). Accumulation of late mRNA's is also regulated at the level of poly (A) site selection. Prior to the onset of DNA synthesis, only LI mRNA's encoding the 52kd and 55kd phosphoproteins accumulate in the cytoplasm. LI pre-mRNA poly (A) sites have additional m-acting elements that are required for recruiting cellular 3’ end processing factors utilised for addition of the 200 residue poly (A) tail (Dezazzo et al., 1991; Imperiale et al., 1995). Late in infection, the situation is reversed and the L2 and L3 poly (A) sites are used 2-3x more frequently than the LI poly(A) site because of a higher affinity for 3' processing factors. Additionally, Larsson
et al. (1992) have shown that by blocking translation late in infection, only LI and L4 mRNA's accumulate in the cytoplasm, implying that the use of the L2, L3, and L5 poly (A) sites require one or more late viral factors.
A further layer of regulation of gene expression is the temporal control of alternative splicing of mRNA transcripts. Most viral mRNA's are matured by removal of one to three introns, the
extreme examples being mRNA for protein IX, which contains no introns, and mRNA encoding the fibre protein, which contains auxiliary x, y and z leaders and requires the removal of six introns (Alestrom et al., 1980). Most often the introns are positioned in the 5' or 3' non-coding portion of the pre-mRNA. For instance, expression of the LI mRNA family represents an example of alternative splicing in which the last intron is spliced using a common 5' splice site and two alternative 3' splice sites, generating mRNA's for the 52kd/55kd (proximal 3' splice site) and Ilia (distal 3' splice site) proteins. The Ilia distal 3' splice site is relatively weak and does not bind cellular splicing factors as effectively as the proximal site. Efficient Ilia splicing requires late viral protein synthesis, possibly virus encoded splicing factors, or virus induced/modified cellular splicing factors (Imperiale et al.,
1995).
Regulation of the E3 splicing system is perhaps the best studied (Scaria and Wold, 1994; reviewed by Wold et al., 1995). The E3 region is embedded within the MLTU which allows for both transcriptional and post-transcriptional control of E3 mRNA. Approximately nine alternatively spliced mRNA are polyadenylated at one of two sites generating two families of mRNA’s. Transcription occurs from the E3 promoter early in infection, but is reduced at late stages, with E3 mRNA arising from MLP activity. These new mRNA contain the tripartite
A
tripartite leader MLTU region u nix I 2 i 3 Ll L2 L3 a dm »—ii ' -.i" —. E1A E1B r>i—> I--- !--- 1--- 1--- 1--- f---H 0 iO 20 30 40 50 60 too HUB. ____1 E2 IVa2
Figure 1.6: Organisation of the Ad2 genome. (A) schematic representation of the MLTU which encodes 5 families of mRNA's (Ll to L5). All mRNA's from this unit receive a common set of 5' leaders through splicing the tripartite leader region. (B) spliced structures of the major mRNA's expressed from the Ll region and their relative expression during infection (reproduced from Nordqvist et al., 1994).
leader sequence and are as abundant as the L4 family of mRNA, thus, the E3 11.6kd protein is a major late protein being detected approximately 24 hours post infection.
All the late mRNA's from the MLTU have a common 201-nucleotide tripartite leader sequence at their 5' end, which is important for cytoplasmic stability and for selective transport late in infection (figure 1.6b). A variant form of this leader contains a 440- nucleotide i-leader exon, the splicing of which is temporally regulated during infection by the E4 ORF3 and E4 ORF6 proteins (Nordqvist et al., 1994; Ohman et al., 1993). E4 ORF3 facilitated splicing of major late mRNA at early times of infection usually leads to the inclusion of the i-leader exon (l,2,i,3), however, the majority of mRNA's expressed late in infection contain the common tripartite leader (1,2,3), which is dependant on the exon skipping functions of E4 ORF6. Interestingly, the functions of E4 ORF3 and ORF6 are dependant on late viral protein synthesis, which if defective, results in late mRNA's retaining the i-leader exon. In addition, The E4 ORF4 inhibition of El A trans-activation of early gene expression, may autoregulate E4 ORF3 and ORF6 splicing events (Bondesson et al., 1996). Viral protein synthesis dominates synthetic activity during the late stages of infection, and cellular protein synthesis ceases. The events involved have been attributed to a variety of processes, for instance, the accumulation of viral mRNA in the cytoplasm is facilitated by the combined functions of the E1B 55kd and E4 ORF6 proteins which do not assist the transport of cellular mRNA's (Babiss et al., 1985; Pilder et al., 1986; Ornelles and Shenk, 1991), although recent evidence indicates that some translocation of small cellular mRNA's via nuclear pore complexes can occur (Smiley et al., 1994). Furthermore, the expression of the 160 nucleotide viral associated mRNA's (VA RNA I and II) blocks the anti-viral activities induced by interferon (Zhang and Schneider, 1993). Interferon activates a double stranded RNA dependant kinase (DAI), which inactivates the essential translation factor elf2-a (O'Malley et al., 1988). This has the effect of blocking the initiation of protein synthesis during viral infection. The VA RNA's are able to prevent this process by binding to the kinase, blocking its activation by interferon, and so allowing the initiation of protein synthesis to proceed. The dephosphorylation of CAP binding protein (elf-4f), another essential translation factor, has been shown to correlate with shutoff of host cell protein synthesis (Yang et al., 1996). Synthesis of viral proteins continues because the common tripartite leader sequence of all viral mRNA's can promote ribosome binding late in infection.
The virus-encoded L4 lOOkd protein is also required for efficient late viral protein synthesis (Riley and Flint, 1993). The protein appears to enhance late translation by binding with mRNA, possibly via the tripartite leader sequence, and may be involved in the selective export of late mRNA's to the cytoplasm.