HRSV and its NS1 and NS2 genes influence the IL-6 and TNF-α gene expression
Although there is much information available on various gene expression during and post HRSV infection, there is scarce data on the sole effects of its NS proteins. Here, we focused on the impact of non-structural proteins NS1 and NS2 on the expression of genes associated with immune response. The expression of genes encoding key pro-inflammatory cytokines (IL-6, TNF-α) and chemotactic cytokines (CCL20 and CXCL8) was measured in the human lung epithelial A549 cells harboring the plasmids encoding NS1 or NS2 proteins. As presented in Fig. 1, the presence of either NS1 and NS2 genes induces the overexpression of TNF-α, whereas the expression of IL-6 was increased in A549 cells harboring the NS2 gene.
It should also be noticed that the control cells transfected with pcDNA3 plasmid did not produce a statistically different response on IL-6 and TNF-α gene expression compared to non-transfected A549 cells. Hence we attribute the overexpression of these genes to NS proteins alone. When HRSV infected the human A549 cells for 150 min., the expression of the IL-6 gene decreased, and the TNF-α not affected compared to non-infected cells (Fig. 1). To integrate these data with data for epithelial cells harboring NS1 or NS2 genes, it should be emphasized that IL-6 and TNF-α gene expression is determined by chromatin remodeling, transcriptional and posttranscriptional mechanisms in which several proteins, microRNAs, and cofactors are involved 12,13. For example, there is the TNF enhanceosome, which is cell type- and stimulus-specific, involving distinct sets of transcription factors and coactivators 12. Thus, non-structural NS1 and NS2 proteins are important, but not the only factor influencing the expression of IL-6 and TNF-α genes. Moreover, during HRSV infection, non-structural proteins have short intracellular half-life; e.g., NS2 protein degrades in the early infection stage within 1–2 hrs 4, and for virus infection, we chose infection time corresponding with the half-life of non-structural proteins. Not to mention, in HRSV-infected cells are other viral proteins, which might together, impact the observed effect.
The expression of CCL20 and CXCL8 was not affected by either gene encoding non-structural HRSV proteins. In turn, after 150 min. HRSV infection, the expression of CCL20 and CXCL8 genes were decreased compared to non-infected cells (Fig. 1).
To sum up, our findings demonstrated that the presence of NS1 and NS2 genes influence the IL-6 and TNF-α gene expression, which might bring us closer to understanding the mechanisms responsible for the cytokine storm, as the robustness of immune response is at odds with inhibition of interferon signaling, a strategy employed by HRSV to enable its replication within the host cell.
Ns1 And Ns2 Genes Of Hrsv Affect The Expression Of Genes Of Ap-1 Transcription Factor
Activator protein 1 (AP-1) transcription factor regulates gene expression in response to various stimuli, including viral infections. AP-1 is a heterodimer composed of Jun and Fos proteins14. We have studied the expression of genes encoding these proteins in human cells A549 with plasmids carrying the NS1 or NS2 genes of HRSV or HRSV-infected cells (Fig. 2).
The presence of NS1 or NS2 genes in the cells and HSRV infection resulted in FOSB subunit deregulation, respectively, up- and down-expression. The results for NS1 and NS2 proteins are in line with data on the E2 protein of human papillomavirus (HPV) and SARS-CoV nucleocapsid proteins, which induce the expression of FOSB and can trigger the AP-1 pathway activation 15,16. Additionally, for the E2 protein of HPV, the ability to induce the expression of FosB in a Bromodomain Protein Brd4-dependent manner was demonstrated. In turn, for non-structural protein 1 (NSP1) of Porcine reproductive and respiratory syndrome virus (PRRSV) the ability for downregulation of FOSB expression was demonstrated 17. However, although Herpes simplex virus type 1 and 2 can activate AP-1 transcription factor, it is not clear which viral protein is responsible for this stimulation 18.
FOS proteins are implicated in the regulation of cell proliferation and differentiation. Interestingly, some studies suggest that FOS proteins can have a pro- or anti-apoptotic effect, depending on subunit type, abundance, active stimuli, and overall composition of AP-1 19. Interestingly, some studies suggest that FOS proteins can have a pro- or anti-apoptotic effect, depending on subunit type, abundance, active stimuli, and overall composition of AP-1. Wu et al. (2013) and Gibbs et al. (2009) demonstrated that HRSV infection resulted in cell cycle arrest in the G0/G1 phase 20. In turn, existing data on cyclin D in HRSV-infected cells are inconsistent. A study performed by Wu et al. (2013) found that there was no change in the levels of D-type cyclins in A549 cells infected with HRSV, while another group reported a decreased gene expression of D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]) 21. Our data also indicates that the expression of cyclin D is unchanged 150 min. post HRSV infection, nor by the presence of NS1 or NS2 genes (Fig. 3).
Corresponding results were obtained for E and p27 cyclin, but not for B cyclin. An essential cell cycle-regulatory molecule, cyclin B controls the cell cycle at the G2/M (mitosis) transition. After 150 min. of HRSV infection cyclin B mRNAs show a decrease in the abundance. The absence of this effect in cells with NS1 or NS2 genes indicates that non-structural proteins separately are not sufficient stimuli to trigger such response, or other viral proteins are engaged in deregulation in cyclin B expression. Although there is no published information on the influence of HRSV infection on B cyclin, it was demonstrated for other DNA and RNA viruses. Herpes Simplex Virus 1 and Minute virus of mice inhibit, whereas Hepatitis C virus and Human papillomaviruses 18 and 31 activate the B cyclin expression 22–25
The NF-κB transcription factor is another protein complex engaged in cellular responses to viral antigens infection 26. Here, we have investigated the expression of NFKB1, the main structural element of the NF-κB complex, and NFKBIA, an inhibitor of NF-κB (IκBα) (Fig. 2). The presence of either NS1 or NS2 genes does not impact the expression of NF-κB genes. 150 min. of HRSV infection also does not deregulate the expression of the NFKB1 gene encoding p50 (Fig. 2). It may be connected with infection time, which in our studies was matched to the short intracellular half-life of NS1 and NS2 proteins. Other data indicate that longer HRSV infection triggers NF-κB, which upon activation, translocates to the nucleus 27,28. Per contra, NS proteins efficiently hijack a couple of pathways that would otherwise conclude with NF-κB activation. It was implicated that the allocation of this transcription factor is due to the abundance of viral RNA detected in the cytoplasm and is not associated with the inhibitory effects of NS proteins 2. It is interesting, as it implies that NS proteins, while having no apparent impact on NF-κB, trigger cytokine expression (e.g., IL-6), through alternative routes.
Although NS proteins and 150 min. HRSV infection does not influence the expression of the NFKB1 gene, the virus at this time down-regulates the expression of NFKBIA, an inhibitor of NF-κB (IκBα). Probably this process is not related to the presence of NS1 or NS2 in the cells (Fig. 2).
To sum up, we found that components of the AP-1 transcription factor are affected by HRSV or its NS1 and/or NS2 proteins. Simultaneously, they do not activate the NF-κB pathway at the early stages of infection. Thus, the activation of cellular pathways can be selective by HRSV, and its proteins and/or infection time may represent a strategy to regulate the cellular signaling process, including host transcriptional machinery, during infection.
Ns1 And Ns2 Genes Induce The Overexpression Of Long Non-coding Rnas, Malat1 And Rp11-510n19.5
As mentioned above, non-coding RNAs (ncRNAs) are indicated as important factors in the response reaction of host cells to pathogens 29. Here, we have studied the expression of long non-coding RNAs: MALAT1, ERICD, and RP11-510N19.5. The first two represent a subtype of lncRNA named lincRNAs, and the latter is a sense intronic.
As presented in Fig. 4, the presence of the NS1 or NS2 genes of HRSV in human cells causes an overexpression of MALAT1 and does not affect the ERICD gene expression. In turn, in the A549 cells with the NS1 gene, the expression of RP11-510N19.5 was increased, and with NS2 decreased.
Thus, for the first time a deregulation of MALAT1 and RP11-510N19.5 lncRNAs by HRSV was shown. Moreover, the effect is triggered not only by complete viral particles but also by the presence of genes of non-structural proteins. So far, it was demonstrated that intact HRSV particle can induce the differential expression of such lncRNAs as MEG3, PVT1, NRAV, and Lnc-n337374, which regulate the host immune response at different steps to inhibit or promote the virus infection, cell cycle regulation and virus replication 30,31.
The influence of HRSV or its NS genes on MALAT1 expression is consistent with results obtained for intact Japanese encephalitis virus (Vellore strain), West Nile virus, Vesicular stomatitis virus, HIV, encephalomyocarditis virus, or herpes simplex virus type 1. However, depending on the specimen, the lncRNA expression is up- or down-regulated 32–34. The differential expression of MALAT1 has also been connected to COVID-19-related inflammation 35.
So far, there is no data on RP11-510N19.5 expression in host cells after viral infection. However, the RP11-510N19.5 gene is located within the gene encoding an ELF3 transcription factor that binds and transactivates ETS (External transcribed spacer) sequences in the promoter of the gene encoding CCL20 chemokine.
Considering these data and the significant engagement of lncRNAs in regulating antiviral host responses, we cannot rule out that MALAT1 and RP11-510N19.5 play such a function in HRSV-infected cells, as MALAT1 has been linked with aiding viral replication and increased inflammation in cells infected by other viruses. And, in the host-virus interplay, viral proteins or genes at early stages of infection may be regulators of the host response by affecting lncRNAs.
To conclude, our results expand the knowledge of molecules participating in regulating the bilateral molecular networks responsible for HRSV-host interactions and point out new, putatively involved mechanisms.