Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors and the process requires the specific recognition of RNA cis-acting signals located in the viral genome. Cellular proteins are involved in coronavirus RNA synthesis together with the p100 transcriptional co-activator protein . A strong interaction between the host cell and the virus replication and transcription processes exists in virus of the Coronaviridae family . In absence of a specific mechanisms for the control of the cell and its replicative machinery for the synthesis of viral products, the viral genome could overcome cellular genes by competition. To overwhelm the cell and focus the transcription on the viral genome, viruses exploits several mechanisms. They can selectively inhibit host cell transcription or eliminate host mRNA and they also can encode their own set of transcriptional activators which facilitate the assembly of the host cell transcription complex at the site of RNA initiation on the viral genome . A virus must counteract intrinsic cellular and innate-immune responses to successfully complete the replication cycle. The viral promoters/enhancers can be activated by the same signalling events as innate-immune genes.
Coronaviruses gives rise to mRNAs that are structurally similar to those of their eukaryotic hosts, and this allows them to parasitize the host machinery to translate the viral mRNA. Specific factors that interfere with host translation or transcription or other factors that enhance viral-specific synthesis are responsible for the increase of virus-encoded molecules .
Recently, it has also been experimentally demonstrated how extensively the host is involved in the SARS-CoV-2 replication / transcription activity. Indeed, more than 500 host proteins constituting the replication/transcription complex (RTC) microenvironment have been identified  . Among these biochemically validated cellular factors, 8 TF that we identified in our analysis were present (STAT1, STAT3, TFEB, NFIX, NFIC, Stat5a, NR3C1,Zfx).
Viral IE control elements might functionally mimic innate immune enhancers, gaining advantage from TF activated by immune signalling to induce viral IE gene expression . Here we found that SARS-CoV-2 genome is enriched in binding sites for TF activated by Interferon-β. Interferons (IFNs), such as IFN-α, IFN-β, and IFN-γ, are important antiviral cytokines released during an infection. These cytokines are activated once the immune cells are faced with an infection. Recent studies have showed that some viruses exploit cis-acting elements and/or TFs related to IFN-induced antiviral activities with the aim of enhancing their replicative processes. In the case of these specific viruses, the IFN response process also facilitates viral replication and gene expression. For example, the genome of human immunodeficiency virus (HIV), a ssRNA virus, contains motifs that are similar to the consensus binding sites for TFs belonging to the host immune genes [33,34].
The HIV promoter has an important role in viral gene expression and several host factors have been demonstrated to contribute to the virus activity and replication. Indeed, HIV core promoter is the central key point for all signals that repress or active viral transcription since it pirates the host cell RNA polymerase II (Pol II) machinery to initiate viral transcription . Coronavirus–host interactions play a crucial role in viral pathogenesis and infection. The coronavirus nonstructural proteins (nsp), in collaboration with recruited host cell proteins, constitute a membrane-associated replication and transcription complexes. SARS-CoV nsp1 was the first CoV nsp1 that was shown to block the expression of reporter gene under the control of IFN-β promoter . From our analysis, a key role of some transcriptional factors such as the family of interferon regulatory factors (IRFs) has emerged. They are a group of transcription factors that are related to the regulation of gene expression and the immune response. In the IRF family, formed by ten members, nine (IRF1–IRF9) have been recognised in mammals, and specifically in humans .
In particular we found two set of TF: one is induced by the TF ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) that drives the first rapid cellular response to the viral infection; the other is induced by the related factor un-phosphorylated ISGF3 (U-ISGF3) that drives the second prolonged response. U-ISGF3 is composed by IFNb-induced IRF9 and STATs 1 and 2 without tyrosine phosphorylation. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). These TF activated by the SARS-CoV-2, support our hypothesis that, mimicking an innate-immune enhancer, they can be directly utilised in a timed manner to guarantee viral gene expression at IE times . Interestingly, our data are also supported by the recent discovery that SARS-CoV-2 Receptor ACE2 is an interferon-stimulated gene and that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2 to enhance infection .
One of the IFN-dependent TF that can bind several domains of the SARS-CoV-2 genome is SIX1. It plays a critical role in embryogenesis, is not expressed in normal adult tissue, but is expressed in many malignancies, including cervical  and HIV-associated lung cancer . However, Hypoxia and HIF-1α can increase SIX1 expression, which reciprocally stimulates HIF-1α expression under both normoxic and hypoxic conditions, thereby creating a positive feedback regulatory loop . Therefore, it is plausible that the hypoxic conditions in SARS-CoV patients contribute to the viral activity by the activation of SIX1 that, in turn, can bind viral ORFs and genes.
Another factor that can bind SARS-CoV-2, HOXA9, has an important role for the C glycoprotein synthesis in the Varicella Zoster Virus. Indeed, PBX / HOXA9 heterodimers bind the promoter region of this viral glycoprotein and HOXA9 homodimer binds the promoter of another viral protein which may upregulate C glycoprotein synthesis .
Moreover, we found a set of TF whose binding sites are present in SARS-CoV- 2 but not in the Bat SARS-like coronavirus despite they have a similarity of 88%.
As shown in our analysis, Interferon Regulatory Factor 4 and 8 (IRF 4 and 8) could be involved with a positive virus interaction. Many RNA viruses are able to induce host translational shut-off, inhibiting the synthesis of peptides and proteins that are necessary for cell response to infection. Many of the RNA viruses, including Caliciviridae, Coronaviridae, Picornaviridae, Oorthomyxoviridae, Reoviridae, and many others, have strategies to induce host translational shut-off and thus prevent the infected cells to synthesize new peptides and proteins, including those IFN-stimulated IRFs and STATs. IRF2 and IRF4 have been implicated in the suppression of type I IFN signaling. [43,44]. IRF-4 and ZNF148 TF are involved in Epstein - Barr virus (EBV) infections. IRF-4 is involved in maintaining the growth phenotypes of EBV-transformed cells in vitro . However, ZNF148 is activated by an early lytic protein encoded by the EBV BMRF1 gene . Therefore SARS-CoV2 could be able to control host translational machinery by IRF-4 and suppress IFN signaling in infected cells.
Interferon regulatory factor 8 (IRF8) is a TF of the IRF family that is induced by interferon in an innate response against infections. This TF is activated by IFN-g in macrophages and stimulates genes essential for a host response. The cellular factor IRF8 facilitates EpsteinBar virus lytic replication by promoting caspase expression and their activation upon lytic induction, in contrast with the most of the IRFs activities that contribute to anti-viral immunity and block the infection or lytic reactivation of herpesviruses [47–50].
Another important TF is the POU Class 4 Homeobox 1 (POU4F2, Brn-3b). It binds in ORF1ab promoter. POU4F2 expression is involved in the activation of immediate-early promoters of herpes simplex virus (HSV) and varicella zoster virus (VZV) leading to virus gene transcription and replication . It is involved also in the transcription of human papilloma virus .The retinoid X receptor α (RXRα) may increase host susceptibility to viral infections by suppressing type I interferon [53,54]. From our network analysis RXRA results linked to the receptor for retinoic acid RARG and to THRB. The overexpression of THRB (TRβ1) and its regulatory effects has been reported to occur during Herpes virus infection (HSV) [55–57]. TRβ1 is a member of the thyroid hormone receptors (TRs) family and it is most abundant in the liver, kidney and the inner ear . In undifferentiated cells, the overexpression of TRβ1 was associated to a remarkably viral replication . Therefore, it would be more interesting to study in the future the interplay between TRβ1 and SARS-COV-2 replication in order to provide important insights into the development of treatments for COVID-19 syndrome.
Additional studies are needed to further validate these findings.
Finally, our functional enrichment analysis showed that several TF, implicated in interferon-mediated response and whose binding sites were specifically found in the sequence of SARS-COV-2 infecting humans, are involved in the regulation of transcription by RNA polymerase II, suggesting a process similar to that of HIV regarding the recruiting of RNA Polimerase II and TFs machinery of the host to initiate viral transcription [59–61].