SARS-CoV-2, like most viruses, relies heavily on host functions to complete its life cycle. It encodes structural proteins (S, E, M and N) that provide a capsule structure for the virion, non-structural proteins (Nsp1-16) that interact with and modify host protein systems for virus transcription and replication, and accessory proteins (Orfs). The latter appear more variable across viruses, even within the same genus, and their functions are less understood; some are required for replication, while others are engaged in transforming the host environment to be more conductive of virus replication and spread. For example, by evading or counteracting the host immune response. Given their highly specialized functions and host interactions, it seems reasonable to assume some viral proteins are more pathogenic than others. Indeed, this has been shown for HIV-1 , ZIKV , SARS-CoV [17, 19, 20, 30, 40] and for SARS-CoV-2 . Toxicity in an in vitro system does not capture the complexity of pathogenicity in a whole organism, nor does it provide any indication as to what tissues might be most infected. Animal studies for SARS-CoV-2 have been limited [2, 7, 12, 15, 29, 39], and few if any have looked at individual proteins. The Drosophila system is well-established and has been instrumental in identifying some of the primary detrimental virus proteins and their interaction with host pathways in HIV-1 and ZIKV infections [3, 11, 14, 21, 25, 26].
Encouraged by the considerable conservation of the SARS-CoV-2 virus-host interaction network proteins between human and fly (Fig. 1), we tested toxicity of individual viral proteins in Drosophila. We generated transgenic fly lines expressing each of the SARS-CoV-2 genes predicted to be most likely pathogenic based on available resources. SARS-CoV-2 Orf6, Nsp6 and Orf7a proofed most pathogenic based on mortality at eclosion (i.e. prior to the adult fly emerges) and longevity (Fig. 2), these were also most toxic in our human in vitro screen using HEK 293 T cells . Nsp3 expression in fly also caused developmental lethality and reduced longevity, albeit with smaller effect than the main pathogenic proteins. Notably, Nsp3 was not found to affect viability of the human cells . This does not exclude possible toxicity to cellular pathways that do not directly affect apoptosis and reflects the added value of looking in a live animal model which captures effects on all cell types and tissues. Orf3a, on the other hand, displayed no detectable change in developmental mortality, however it significantly reduced fly lifespan and showed cytotoxicity in HEK 293 T . These data suggest that in fly, Orf3a either induced relatively moderate toxicity or interacts with, and affects, host systems that play a role after adult hatching. Taken together, these findings show the fly system can accurately capture SARS-CoV-2 individual protein pathogenicity.
Similar to host tissue-specific effects based on tissue differences in proteins and pathways, the individual virus proteins are equally important in determining the effects on a tissue. Our data revealed similar phenotypic read-outs for SARS-CoV-2 Orf6, Nsp6 and Orf7a expression across the various tissues in fly, however, the underlying pathomechanisms are likely different. Based on available literature for SARS-CoV-2 and related SARS-CoV proteins, we can make some predictions as to the pathways affected by each. Orf6 has been known to have interferon (IFN) antagonistic properties based on previous coronaviruses. This function has been retained by SARS-CoV-2 Orf6 [23, 24], with ability to interfere with interferon production comparable to its SARS-CoV equivalent . SARS-CoV Orf6 has been shown to inhibit primary interferon production  and to antagonize STAT1 function (interferon signaling) by altering the host nuclear import factors . Indeed, interaction of SARS-CoV-2 Orf6 with proteins of the nuclear pore complex  and its effect on IFN stimulated genes (ISG)  have been reported recently. The interaction with the nuclear pore complex is evident from our data as well (Selinexor; Fig. 6). SARS-CoV Nsp6 localizes to the endoplasmic reticulum, where it has been shown to interact with Nsp3 and Nsp4 to induce double-membrane vesicles , and its SARS-CoV-2 equivalent has been predicted to interact with multiple ATPases of vesicle trafficking . It might also interact with SIGMA1R, a receptor thought to regulate the endoplasmic reticulum stress response . SARS-CoV-2 Orf7a protein has been predicted to interact with ribosomal transport proteins HEATR3 and MDN1 . Previously, SARS-CoV Orf7a has been demonstrated to inhibit cellular translation and induce apoptosis , in line with our viability data for SARS-CoV-2 Orf7a. The SARS-CoV protein was shown to interact with Bcl-XL and other pro-survival proteins (Bcl-2, Bcl-2, Mcl-1, and A1), but not with pro-apoptotic proteins (Bax, Bak, Bad, and Bid) . The findings suggested Orf7a might trigger apoptosis by interfering directly with the pro-survival function of Bcl-XL, indeed both proteins were shown to co-localize at the endoplasmic reticulum and mitochondria . Together, these findings indicate that even though the SARS-CoV-2 Orf6, Nsp6 and Orf7a proteins lead to similar viability defects and tissue damage, the underlying pathomechanisms are likely different.
SARS-CoV-2 Orf6 has been shown to act as an antagonist of IFN signaling [23, 24, 27, 41] as well as to directly interact with the NUP98-RAE1 complex at the nuclear pore [10, 23, 24, 27]. We similarly report SARS-CoV-2 Orf6 protein–protein interaction with many key members of the nuclear pore machinery, including nucleoporins and karyopherins (both importins and exportins) . Selinexor, an FDA-approved selective inhibitor of nuclear export , has been predicted to disrupt this interaction of SARS-CoV-2 Orf6 with the host nuclear pore complex . We report it reduced SARS-CoV-2 Orf6 cytotoxic effects in a human in vitro culture model . Notably, the nuclear pore proteins in the Orf6 interaction network are extremely conserved between human and fly (Fig. 1) , therefore we similarly treated the flies with this nuclear export inhibitor. Indeed, Selinexor treatment greatly attenuated the Orf6-induced muscle and trachea (lung) defects observed in fly (Figs. 5 and 6). However, even though we found phenotypic overlap between Orf6, Orf7a and Nsp6 in our assays, Selinexor was unable to counteract Orf7a and Nsp6 effects, supporting the notion that the viral proteins act through different pathomechanisms. These findings further underpin our hypothesis that inhibition of viral entry/replication (the strategy of COVID-19 interventions like Remdesivir) might proof insufficient by itself as the approach might stop the virus in its tracks but does not remedy the multi-tissue effects of virus already present in the host/pateint. Therefore, we propose to use our Orf6 findings as a starting point to identify additional virus-host pathogenic interactions, then to develop drugs to target and disrupt these interactions. Therapeutic strategies, combining inhibition of virus entry/replication and pathogenic host interactions, would both stop SARS-CoV-2 spread and ameliorate the devastating progression of COVID-19 symptomatology.
In the current study, we have identified SARS-CoV-2 primary determinant pathogenic proteins (Orf6, Nsp6 and Orf7a). Capitalizing on the whole fly, enabled us to study the pathogenic effects of the individual viral proteins on muscle and trachea (lung). We expanded our understanding of the pathomechanism invoked by Orf6 and showed that Selinexor was able to attenuate Orf6 pathogenicity specifically in vivo. Together with the high conservation across the SARS-CoV-2 virus-host protein interaction network between human and fly, these data strongly support the fly as an effective model system for studying individual SARS-CoV-2 proteins.