Covid-19 and virus: gene MHC class II transactivator (CIITA) induces resistance in human cell lines

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Discoveries from the Benaroya Research Institute at Virginia Mason (BRI) have identified a new cellular protection pathway that targets a common vulnerability in several different pandemic viruses, and collaborators at Case Western Reserve University, Boston University School of Medicine and MRIGlobal have shown that this pathway can protect cells from infection by Ebola virus and coronaviruses, like SARS-CoV-2.

Published today in Science, these new findings provide a better understanding of cellular mechanisms involved in viral resistance that can inform future treatments and therapies for viral infectious diseases.

The research illuminates a completely new role for the two genes identified and a unique approach to inhibiting virus fusion and entry into human cells – getting us one step closer to the next generation of antiviral therapies. Researchers used a transposon-mediated gene-activation screen to search for new genes that can prevent infection by Ebola virus.

This new screening strategy – that serves as a blueprint for uncovering resistance mechanism against other dangerous pathogens – found that the gene MHC class II transactivator (CIITA) induces resistance in human cell lines by activating the expression of a second gene, CD74.

One form of CD74, known as p41, disrupts the processing of proteins on the coat of the Ebola virus protein by cellular proteases called Cathepsins.

This prevents entry of the virus into the cell and infection. CD74 p41 also blocked the Cathepsin-dependent entry pathway of coronaviruses, including SARS-CoV-2.

“Uncovering these new cellular protection pathways is incredibly important for understanding how we disrupt or change the virus infection cycle to illicit better protection against viruses like Ebola or SARS-CoV-2,” said Adam Lacy-Hulbert, Ph.D., Principal Investigator, BRI and lead author on the study. “And our new strategy helps us find mechanisms that have eluded conventional genetic screens.”

The findings illustrate a new role for genes previously thought to be involved in more conventional T cell and B cell mediated immune responses.

For example, CIITA was understood as important for communication between immune cells, but it had not previously been seen as a way for cells to defend themselves against viruses.

“As a virologist, I am excited not just about what this means for Ebola virus, but about the broader implications for other viruses,” said Anna Bruchez, Ph.D., Instructor in Pathology, Case Western Reserve University and co-author on the study.

“Many viruses, including coronaviruses, use cathepsin proteases to help them infect cells. Fortunately, when SARS-CoV-2 emerged, I had recently moved to Case Western, and was able to use their specialized BSL3 laboratories to show the CD74 pathway also blocked endosomal entry by this virus.

Thus, this anti-viral mechanism has evolved to work against many different viruses.”

“We really don’t understand the cellular mechanisms that block viral infections which has limited our ability to effectively respond to pandemics, including this year’s coronavirus,” said Lynda M. Stuart, M.D., Ph.D., Deputy Director, Bill & Melinda Gates Foundation, BRI Affiliate Investigator and co-author on the study.

“We really need therapies that can block all viruses, including unknown future pathogens.

To do that we need to find common pathways that viruses target and then develop approaches to block those vulnerabilities.

Our work demonstrates one way in which cells can be modified to do this, and we hope that our insights will open up new avenues for scientists developing therapies and interventions to treat viral infectious diseases that impact millions of lives around the world.”


HCoV-EMC was isolated from a patient who died from an acute respiratory disease similar to that caused by SARS-CoV. However, there are several indicators that the host responses to these two viruses may be significantly different. Several cases of HCoV-EMC infection have resulted in renal failure, which has rarely been ob- served in SARS-CoV infection.

In addition, SARS-CoV and HCoV-EMC do not use the same cell receptor, and there are im- portant differences in their genomic sequences. This study adds strength to the assertion that “HCoV-EMC is not the same as SARS-CoV” (23).

Indeed, even though we identified specific char- acteristics of the SARS-CoV response in the HCoV-EMC signa- tures, HCoV-EMC induced robust and specific transcriptional re- sponses that were distinct from those induced by SARS-CoV, including the broad down-regulation of MHC molecules.

This study is the first global transcriptomic analysis of the cel- lular response to HCoV-EMC infection. Kindler et al. performed RNA-Seq on human airway epithelium (HAE) cells infected with HCoV-EMC (24).

However, their analysis was focused on viral sequences and did not include a genome-wide analysis of the host response. They did, however, use RT-qPCR (quantitative PCR) to compare expression levels of a set of 15 genes, including IFN, RNA sensor molecules, and IFN-stimulated genes (ISGs), following in- fection with HCoV-EMC, SARS-CoV, or HCoV-229E (MOI 0.1).

In our study, we confirm that SARS-CoV and HCoV-EMC induce a similar up-regulation of RNA sensor molecules, such as RIGI, MDA5, and two of three genes of ISGF3 (IRF9 and STAT1) (genes in cluster I [Fig. 3]).

Of note, HCoV-EMC titers were up to 102- fold higher than those of SARS-CoV in HAE cells (24), whereas we observed similar viral replication of the two CoVs in Calu-3 cells. Lower replication of SARS-CoV in HAE cells might be explained by the mixed cell population in these primary cultures, with likely nonuniform expression of SARS-CoV receptor (ACE2).

In con- trast, Calu-3 2B4 cells used in our study are a clonal population of Calu-3 cells sorted for ACE2 expression which support high rep- lication of SARS-CoV. In addition, while Kindler et al. noted the absence of induction of IFN at 3, 6, and 12 hpi (24), we found a specific up-regulation of IFNa5 and IFN– β 1 by HCoV-EMC at 18 and 24 hpi (genes in cluster III) and an up-regulation of IFNa21 by both SARS-CoV and HCoV-EMC at 24 hpi (cluster I) (expression values for all DE genes are available at http://www.systemsvirology.org).

These data illustrate that HCoV-EMC and SARS-CoV both trigger the activation of pattern recognition re- ceptors but may subsequently induce different levels of IFN. Moreover, there were stark differences in global downstream ISG expression following infection with SARS-CoV or HCoV-EMC; this analysis is discussed in detail elsewhere (V. D. Menachery et al., submitted for publication).

Activation of similar innate viral-sensing pathways by HCoV- EMC and SARS-CoV is not surprising given the conservation of this mechanism to detect foreign RNA and familial relationships of the viruses. We also found that both viruses induced proinflam- matory cytokines related to IL-17 pathways.

It has previously been shown that IL-17A-related gene expression exacerbates severe re- spiratory syncytial virus (RSV) or influenza virus infection (25, 26).

IL-17A was predicted to be activated throughout infection with HCoV-EMC and may induce immune-mediated pathology that possibly contributes to a high mortality rate. IL-17A is known to be produced by T-helper cells, but its expression in Calu3 cells was increased up to 2-fold at 24 hpi after HCoV-EMC infection.

Interestingly, IL-17C and IL-17F, which can be produced by epi- thelial cells under certain inflammatory conditions and which ac- tivate pathways similar to IL-17A-mediated responses (27), were increased earlier and to a greater extent following HCoV-EMC infection (up to 3-fold at 18 hpi for IL-17C and 4-fold at 7 hpi for IL-17F).

Therefore, further study of the IL-17 response may pro- vide interesting targets to limit lung injury (26).

A main difference between responses to HCoV-EMC and SARS-CoV was the specific down-regulation of the antigen pre- sentation pathway after HCoV-EMC infection.

In contrast, these genes were found to be up-regulated after SARS-CoV infection. Several viruses have evolved mechanisms to inhibit both the MHC class I (reviewed in references 28 and 29) and class II (reviewed in reference 30) pathways. While expression of MHC class II is usu- ally limited to professional antigen-presenting cells, human lung epithelial cells constitutively express this complex (31).

Our data demonstrated down-regulation of the MHC class II transactivator (CIITA) after HCoV-EMC infection, a finding that possibly ex- plains decreases in MHC class II molecule expression; this is a common viral strategy used to block that pathway (30).

MHC class II inhibition can prevent class II-mediated presentation of endogenous viral antigens produced within infected cells and im- pair the adaptive immune response. Similarly, MHC class I genes were also down-regulated after HCoV-EMC infection; decreasing expression of MHC class I can attenuate CD8 T-cell-mediated recognition of infected cells and could allow immune evasion by HCoV-EMC. Finally, PSMB8 and PSMB9, parts of the immuno- proteasome, were also down-regulated by HCoV-EMC; these components replace portions of the standard proteasome and en- hance production of MHC class I binding peptides (32).

In their absence, proteins targeted for degradation may not generate pep- tides that robustly bind MHC class I, thus limiting their presenta- tion. Down-regulation of PSMB8 and PSMB9 could counteract the host response to viral infection, including up-regulation of ubiquitins and ubiquitin ligases observed during HCoV-EMC infection (Fig. 3B) that may ineffectively target viral protein for deg- radation. Together, the inhibition of MHC class I and II as well as immunoproteasome construction may have an important impact on the in vivo adaptive immune response against HCoV-EMC.

While there is no proven effective antiviral therapy against SARS-CoV (33), several molecules have in vitro antiviral activity, including ribavirin, lopinavir, and type I IFN, but their benefits for patients are unclear (33).

IFN-a pretreatment of cells has been shown to inhibit HCoV-EMC replication (24), but no direct an- tiviral therapies have been reported. Targeting host factors important for the virus, instead of the virus itself, has been investigated for HIV (34) and influenza virus (13).

For example, inhibiting upstream regulators (such as NF-KB) that control the host re- sponse to influenza virus infection has been shown to reduce virus replication in vitro and in mice (35).

Inhibition of immunophilins that interact with the viral nonstructural protein 1 (Nsp1) resulted in potent inhibition of SARS-CoV replication (36, 37).

In this study, we characterized upstream regulators predicted to be acti- vated (e.g., NF-KB and IL-17, which could be targeted with spe- cific inhibitors) and upstream regulators predicted to be inhib- ited.

The top five inhibited regulators included one glucocorticoid and four kinase inhibitors; these drugs may be able to directly block part of the host response and impact viral replication/patho-genesis. Among them, LY294002, a potent inhibitor of phospha- tidylinositol 3 kinase (PI3K), has known antiviral activity, inhibiting the replication of influenza virus (38), vaccinia virus (39), and HCMV (40).

SB203580, an inhibitor of p38 MAPK, is also an effective antiviral against the encephalomyocarditis virus (41), RSV (42), and HIV (43). LY294002 and SB203580 were also iden- tified in Connectivity Map, a database of drug-associated gene expression profiles (22), as molecules reversing components of the HCoV-EMC gene expression signature. Finally, SB203580 showed promising antiviral results against both HCoV-EMC and SARS-CoV in our in vitro assay (Fig. 4C).

Further extensive studIes, including dose-response tests and tests of other kinases inhib- itors, are ongoing. Nonetheless, these results validate our genome-based drug prediction, which allows rapid identification of effective antivirals. Despite central roles of PI3K and MAPK pathways in regulating multiple cellular processes, many kinase inhibitors targeting these pathways have been shown to be safe and well tolerated in vivo (reviewed in references 44 and 45).

It has been hypothesized that mitogenic MAPK and survival PI3K/Akt pathways may be of major importance only during early development of an organism and may be dispensable in adult tissues (13).

Several drugs targeting JNK, PI3K, and MEK have shown promising therapeutic potential in humans against a variety of diseases, including cancer and inflammatory disorder (44, 45). p38 MAPK inhibitors have also been evaluated in humans, but the first gen- eration of molecules, including SB203580, has a high in vivo toxicity (liver and/or central nervous system).

However, develop- ment of novel nontoxic inhibitors (e.g., ML3403) (46), more selective molecules (e.g., AS1940477) (47), and administration via inhalation (48) are promising strategies for use of this class of inhibitor for treatment of pulmonary disease. Overall, these results indicate that kinase inhibitors could be used as broad anti-CoV agents which might be combined with other host-targeting molecules, like peroxisome proliferator-activated receptor a (PPARa) agonists, to better inhibit HCoV-EMC replication.

In conclusion, using global gene expression profiling, we haveshown that HCoV-EMC induces a dramatic host transcriptional response, most of which does not overlap the response induced by SARS-CoV. This study highlights the advantages of high- throughput “-omics” to globally and efficiently characterize emerging pathogens.

The robust host gene expression analysis of HCoV-EMC infection provides a plethora of data to mine for further hypotheses and understanding. Host response profiles can also be used to quickly identify possible treatment strategies, and we anticipate that host transcriptional profiling will become a general strategy for the rapid characterization of future emerging viruses.

REFERENCE : DOI: 10.1128/mBio.00165-13


More information: MHC class II transactivator CIITA induces cell resistance to Ebola virus and SARS-like coronaviruses, Science (2020). DOI: 10.1126/science.abb3753 , science.sciencemag.org/content … 8/26/science.abb3753

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