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The HRSV genome contains a single promoter located in the leader region which controls RNA synthesis (Dickens et al., 1984). For the RNA synthesis in transcription or replication, two different models are hypothesised. In the first model, transcription and replication start at the same site of the leader sequence. Having been transcribed, the RNA faces three possibilities: 1. the synthesis of an immature RNA that dissociates from the template near the leader region, 2. RNA being packaged into the N protein and forms a stable complex which results in the synthesis of cRNA, and 3. RNA associates with the N protein and makes a stable complex, but RNA elongation ends at the leader region and reinitiates at the gene start region. In the second model, it is proposed that transcription and replication start at distinct but overlapping sites of the leader regions. Nucleotides 3C, 5C, 8U, 9U, 10U, and 11U of the leader sequence are the common elements of the two promoters (see Section 1.2.2 for description on the leader sequence). Nucleotides 1U, 2G, 6U, and 7U of the leader sequence are involved in the recognition of the RNP complex for replication complex and the gene start

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signals for the transcription initiation (Fearns et al., 2002). There are other mechanisms proposed for transcription initiation which are reviewed by Cowton et al. (2006).

It has been suggested that the transcriptional complex having been bound to the leader region, starts scanning the genome for the gene start sequence and initiates mRNA synthesis from the NS1 gene (Cowton & Fearns, 2005). The cis factor for termination of gene transcription is the gene end sequence (Section 1.2.2) (Harmon et al., 2001). It has been shown that if the polymerase does not recognise the gene end signal, it will result in the production of a polycistronic RNA containing the intergenic junction (Dickens et al., 1984).

One of the fundamental differences between the genomic structure of PVM and HRSV is the overlap of the M2 and L genes seen in HRSV. The L gene shares its 5’ sequence with 3’ end of M2 gene. In PVM, however, both M2 and L genes form separate and distinctive genes on the genome (Figure 1.1) (Collins et al., 1987).

1.2.4.1 Gene expression gradient

Early reports indicated that after termination in transcription, the RNP complex dissociates from the cRNA templates and a proportion of the RNP complex restart transcription by relocating to the promoter sequence in the 3’ end of vRNA. The remaining proportion continues transcription of the next gene (Dickens et al., 1984). This stop-start process generates a gradient of transcription with mRNAs representing leader proximal genes being more abundant than those from leader-distal genes. The gradient of expression of HRSV genes was confirmed by Barik (1992) in which the concentration of individual viral mRNA molecules were calculated using a slot blot assay and the molar ratio of mRNA was calculated against the NS1 mRNA. The percentage of mRNA for each gene of RSV is shown in Table 1.6.

Gene NS1 NS2 N P M SH G F M2-1 L

Molar ratio 100 95 90 68 52 32 21 18 15 3

Table 1.6 Percentage molar ratio of HRSV mRNA indicating the genome expression gradient

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1.2.4.2 mRNA capping

Neither the vRNA nor cRNA contain a 5’ cap structure. However, mRNA molecules are capped (m7G(5')ppp(5')Gp) and polyadenylated (Barik, 1993). Barik (1993), using radioactive S-adenosyl-methionine, showed that the methyl group, but not the G residues, is added by host enzymes. In the same study it was shown that both the cap structure formation and the cap methylation are coupled to transcription and on- going transcription is required for cap formation and methylation and that the cap itself does not have any effect on transcription.

More recently, it has been reported that the L protein of vesicular stomatitis virus (VSV), a member of family Rhabdoviridae within the order Mononegavirales, is responsible for capping of pre-mRNA molecules (Li et al., 2006), and specifically that the region V of the L protein is responsible in the pre-mRNA capping process (Li et al., 2008b). Mutational analysis has shown that the capping process happens not in the conventional way of mRNA capping, but in a unique way using histidine residue in the V domain of the L protein (known as the HR motif) (Ogino et al., 2010). It is likely that a similar process occurs in pneumoviruses with the L protein carrying out the capping process. The proposed mechanism for capping mRNA is shown in Figure 1.7 in comparison with the normal cellular capping process.

Figure 1.7 The process involving cap synthesis in eukaryotes and in VSV. A. In eukaryotic cells the γ phosphate of pre-mRNA is removed by the action of RNA 5′-triphosphatase (RTPase) (1). In the next steps (2 and 3) a GMP is transferred by a guanylyltransferase (GTase) enzyme action to the pre-mRNA to produce the 5’ cap structure. B. In VSV the L protein mediates both reactions and removes two phosphate groups from the tri-phosphate-pre-mRNA structure (1a) and transfers a GDP to the pre-mRNA structure by its RNA:GDP polyribonucleotidyltransferase (PRNTase) activity (2a and 3a). Adapted from Ogino et al.(2010).

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1.2.4.3 Transcription termination and polyadenylation

Harmon and colleagues (2001) have summarised the cis factors involving in the transcription termination in three categories: the two conserved regions of the Gene End sequence (the 3’-UCAAU-5’ and the U track) and the central region which is located between the conserved regions (Section 1.2.2). Analysis of the gene end sequence of the M gene of HRSV vRNA has shown that nucleotides at positions 2 to 6 are important for transcription termination, whereas the nucleotides at positions 1 and 7 are not important to obtain efficient transcription termination. At position 8 of the gene end sequence, an A or U residue allows termination. The presence of four U residues is necessary for efficient transcription termination, and genes of HRSV with shorter U residues have been shown to fail to produce mRNA efficiently and produce readthrough polycistronic RNA templates (Harmon et al., 2001). Poly-adenylation occurs when the polymerase complex reaches to the gene end sequence. The presence of a U rich sequence in the gene end directs the RNP complex to generate the poly A sequence.

In contrast with the gene start sequence, the gene end sequence is not well conserved among the pneumoviruses. In PVM the gene end sequence consists of uAGUuAnnn(A)n and in HRSV it consists of AGU(U/A)Annnn(A)n (Chambers et al., 1991; Melero, 2006). The gene end sequence is followed by the non-conserved intergenic sequence (Chambers et al., 1991). It was shown that the length or structure of the intergenic sequence does not affect the termination of transcription (Kuo et al., 1996). This finding was challenged with another report describing the importance of the M/SH gene junction variation in the expression of the SH (Moudy et al., 2004). Moudy and colleagues (2004) reported the P/M gene junction as the most variable gene junction with no detectable variation in the gene expression.