El principio de veracidad

Interferon-stimulated genes modulate biologic effects of IFNs. There are >300 ISGs which can be assigned to functional categories. The specific function of a large proportion of these is unclear, but some are known to play key roles in host defence (272). Below I outline some of the highly differentially transcribed ISGs within my dataset and highlight those that are particularly pertinent to RSV infection.

6.6.5.1 Ubiquitin Specific Peptidase 18

Ubiquitin Specific Peptidase 18 (USP18), also known as ISG43, was the highest upregulated mRNA at the early time point (FC 7.95). This protein cleaves ubiquitin from ubiquitinylated substrates, specifically ISG15 fusions. ISG15 is a ubiquitin-like protein that is conjugated to intracellular target proteins (ISGylation) upon activation by IFN. ISGylation may modulate the JAK-STAT pathway. The secreted form of ISG15 has a number of roles; it can induce natural killer cell proliferation, act as a chemotactic factor for neutrophils and as an IFN-γ-inducing cytokine. The ISG15/USP18 pathway has been found highly expressed in response to a number of viruses, including hepatitis C and vesicular stomatitis virus (273, 274).

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Figure 6-10 Interferon signalling pathway through Jak-STAT pathway

The cell response to IFN can be mediated through the Jak-Stat pathway. Activation of Jak kinases and phosphorylation of STAT1 allows association with IRF-9 to form ISGF3 transcription factor, which enters the nucleus and drives the cell into an antiviral state. Downstream IFI35, IFIT1, MX1 and IFIT 3 were all transcriptionally upregulated in the microarray data.

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6.6.5.2 Myxovirus resistance 1

Myxovirus Resistance 1 (MX1) gene encodes a GTP-metabolising protein that participates in the cellular antiviral response. It is activated through the Jak-STAT pathway, induced by type I and II IFNs and antagonises the replication process of RNA (particularly negative stranded) and DNA viruses. It achieves this by binding and inactivating their ribonucleocapsid. MX genes were discovered around 50 years ago by Lindenmann et al (275). The protein encoded by MX1, called MXA, has a broad antiviral activity, which has been studied in the context of infection with a large number of different RNA viruses (276). In measles, a member of the paramyxoviridae family, MX1 has been shown to inhibit virus in a human mononuclear cell line (277). In contrast RSV infection of Vero cells was resistant to the antiviral effects of MxA (278). In a study using RSV infected cotton rats MX1 and 2 were highly expressed in the lungs early in infection and in a dose dependant fashion, which the authors concluded, was evidence of a robust type 1 IFN response (279).

6.6.5.3 Tetherin

Tetherin, encoded by the BST2 gene, is expressed as part of the IFN dependant antiviral response pathway and is a viral ‘restriction’ factor. This was significantly upregulated at the early time point. A mechanism whereby tetherin inhibits enveloped virus release by tethering the budding virus like particles has been proposed (280). Tetherin has been associated with all tested retroviruses, and some filoviruses. It has been recently reported that tetherin can act as a PRR inducing NF-kB dependant pro-inflammatory gene expression in virus infected cells (281).

6.6.5.4 Double stranded RNA-specific endoribonuclease

Double stranded RNA-specific endoribonuclease (DICER) gene encodes a protein that possesses an RNA helicase motif containing a DEXH box. This ISG was down-regulated by RSV exposure, a finding confirmed by PCR. It functions as a ribonuclease and plays a central role in short dsRNA- mediated post-transcriptional gene silencing. Gene silencing, mediated by RNA inference, controls the elimination of transcripts from mobile and

169 repetitive DNA elements of the genome, and the degradation of exogenous RNA of viral origin. Reduced DICER expression in the cord blood of infants subsequently admitted with severe RSV disease has recently been reported. The authors hypothesized that this reduction might have led to diminished anti-viral activity and impairment of innate cellular function (282). They did not measure the level of DICER in the patients at the time of illness. It is possible that those children who are already predisposed by low DICER levels and thus a reduced ability to interfere with viral replication are made more so by RSV mediated down regulation of DICER.

A number of groups of genes, whose antiviral functions have previously been recognised, were well represented in the data, including the APOBEC3, TRIM and IFIT families. The mechanism by which many of these genes inhibit viral replication is not fully known, but it is possible that they interfere with one or more of the following: viral entry, viral DNA synthesis, intracellular movement of viral nucleic acids and viral gene expression (283- 285).

6.6.5.5 APOBEC3 enzymes

APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide- like) is a superfamily of enzymes encoded by the APOBEC3 gene. They are capable of deaminating cytidines in single stranded DNA replication intermediates, leading to cytidine being changed to uridine and guanine to adenine, a phenomenon termed DNA editing. APOBEC3G is the most widely studied member of the family. In my dataset, APOBEC3 -A, -B, -C, -F and -G were upregulated, with APOBEC3-F and –G being the most highly expressed. RNA virus replication of measles, mumps and RSV, has been found to be sensitive to A3G (APOBEC3G) inhibition in in vitro study (284).

6.6.5.6 TRIM Proteins

A number of genes encoding proteins of the TRIM (tripartite motif-containing proteins) family were differentially expressed within my dataset. Thus TRIM 5, 14, 21, 26, 34 and 56 were up-regulated and TRIM 8 and 11, down- regulated. The TRIM family is a diverse family of RING finger domain-

170 containing protein that are classified into 11 subgroups. They are involved in the regulation of innate immune responses through modulation of PRR signalling pathways including TLRs and RIG-I. Many TRIM proteins are induced by type I IFN stimulation (286, 287). TRIM 21 has previously been shown to be induced by RNA viral infection, and is an essential modulator of IRF3 stability (288). TRIM proteins can exert antiviral activity through modulation of viral proteins as well as interacting with the innate immune signalling pathways (289). TRIM 56 was recently identified to associate with the N-terminal protease of bovine diarrhoea virus and inhibit its replication (285). TRIM 5, 11, 26 and 31 have been demonstrated to inhibit HIV entry. TRIM 11, which was down-regulated in the analysis, inhibits RIG-1 mediated IFN beta production by targeting the TBK1 complex (290).

6.6.5.7 IFIT protein encoding genes

IFIT (interferon-induced protein with tetratricopeptide repeats) protein encoding genes were over-represented at 4 hours, all by a >3 fold change, specifically –1, -1B, -2, -3, -5. Their transcription can be induced by viral infection. IFIT proteins show antiviral activity in two ways. They bind to both viral nucleic acids and to eIF3 (eukaryotic initiation factor 3) thereby inhibiting the translation process (283). IFIT1-5 genes were expressed in the whole blood analysis of infants with RSV (255).