The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7-Cyclin H Determines Individual Patterns of Transcription in Infected Cells.

The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.

Results of Tick-Borne Encephalitis Virus (TBEV) Diagnostics in an Endemic Area in Southern Germany, 2007 to 2022.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted neurotropic flavivirus in Europe and Asia. Our analysis aimed to investigate the contribution of TBEV-specific antibody detection by serological assays and TBEV RNA detection by real-time PCR to the diagnosis of tick-borne encephalitis (TBE). We analyzed data from 3713 patients from 16 years of laboratory TBEV diagnostics in an endemic area in Southern Germany. During this period, 126 cases of TBE were diagnosed. TBEV-specific IgM ELISA tests showed a high clinical sensitivity (96.8%) and a very high clinical specificity (99.7%). In immunocompetent patients, TBE was reliably diagnosed by detection of TBEV IgM antibodies in serum. Intrathecal TBEV IgG antibody synthesis was detected in 46 of 84 (55%) cases by analysis of paired serum and cerebrospinal fluid (CSF) samples. None of the 87 immunocompetent TBE patients tested had detectable TBEV RNA in serum or CSF. In contrast, in two TBE patients without TBEV-specific antibodies, diagnosis could only be made by the detection of TBEV RNA in CSF. Both patients had previously been treated with the B cell-depleting antibody rituximab. Therefore, in patients with CNS infection and humoral immunodeficiency, it is necessary to include TBEV PCR in the diagnostic approach.

HIV-1 neutralizing antibodies provide sterilizing immunity by blocking infection of the first cells.

Neutralizing antibodies targeting HIV-1 Env have been shown to protect from systemic infection. To explore whether these antibodies can inhibit infection of the first cells, challenge viruses based on simian immunodeficiency virus (SIV) were developed that use HIV-1 Env for entry into target cells during the first replication cycle, but then switch to SIV Env usage. Antibodies binding to Env of HIV-1, but not SIV, block HIV-1 Env-mediated infection events after rectal exposure of non-human primates to the switching challenge virus. After natural exposure to HIV-1, such a reduction of the number of first infection events should be sufficient to provide sterilizing immunity in the strictest sense in most of the exposed individuals. Since blocking infection of the first cells avoids the formation of latently infected cells and reduces the risk of emergence of antibody-resistant mutants, it may be the best mode of protection.

ORF3c is expressed in SARS-CoV-2-infected cells and inhibits innate sensing by targeting MAVS.

Most SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While the majority of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. One example is the ORF3a sgRNA that also encodes ORF3c, an enigmatic 41-amino-acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2-infected cells and suppresses RIG-I- and MDA5-mediated IFN-ß induction. ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage, and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c, demonstrating that this reading frame is not essential for efficient viral replication in vivo and is likely compensated by other viral proteins. In agreement with this, disruption of ORF3c does not significantly affect SARS-CoV-2 replication in CaCo-2, CaLu-3, or Rhinolophus alcyone cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.

Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria.

Human inherited disorders of interferon-gamma (IFN-γ) immunity underlie severe mycobacterial diseases. We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to interleukin-23 (IL-23) and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN-γ by innate-like adaptive mucosal-associated invariant T cells (MAIT) and γδ T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-γ.

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is critical for infection in a cell-specific manner and functions at the attachment step on keratinocytes.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several B cell malignancies and Kaposi's sarcoma. We analyzed the function of K8.1, the major antigenic component of the KSHV virion in the infection of different cells. To do this, we deleted K8.1 from the viral genome. It was found that K8.1 is critical for the infection of certain epithelial cells, e.g., a skin model cell line but not for infection of many other cells. K8.1 was found to mediate attachment of the virus to cells where it plays a role in infection. In contrast, we did not find K8.1 or a related protein from a closely related monkey virus to activate fusion of the viral and cellular membranes, at least not under the conditions tested. These findings suggest that K8.1 functions in a highly cell-specific manner during KSHV entry, playing a crucial role in the attachment of KSHV to, e.g., skin epithelial cells.

SARS-CoV-2 variants with NSP12 P323L/G671S mutations display enhanced virus replication in ferret upper airways and higher transmissibility.

With the emergence of multiple predominant SARS-CoV-2 variants, it becomes important to have a comprehensive assessment of their viral fitness and transmissibility. Here, we demonstrate that natural temperature differences between the upper (33°C) and lower (37°C) respiratory tract have profound effects on SARS-CoV-2 replication and transmissibility. Specifically, SARS-CoV-2 variants containing the NSP12 mutations P323L or P323L/G671S exhibit enhanced RNA-dependent RNA polymerase (RdRp) activity at 33°C compared with 37°C and high transmissibility. Molecular dynamics simulations and microscale thermophoresis demonstrate that the NSP12 P323L and P323L/G671S mutations stabilize the NSP12-NSP7-NSP8 complex through hydrophobic effects, leading to increased viral RdRp activity. Furthermore, competitive transmissibility assay reveals that reverse genetic (RG)-P323L or RG-P323L/G671S NSP12 outcompetes RG-WT (wild-type) NSP12 for replication in the upper respiratory tract, allowing markedly rapid transmissibility. This suggests that NSP12 P323L or P323L/G671S mutation of SARS-CoV-2 is associated with increased RdRp complex stability and enzymatic activity, promoting efficient transmissibility.

A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity.

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.

Re-Analysis of the Widely Used Recombinant Murine Cytomegalovirus MCMV-m157luc Derived from the Bacmid pSM3fr Confirms Its Hybrid Nature.

Murine cytomegalovirus (MCMV), and, in particular, recombinant virus derived from MCMV-bacmid pSM3fr, is widely used as the small animal infection model for human cytomegalovirus (HCMV). We sequenced the complete genomes of MCMV strains and recombinants for quality control. However, we noticed deviances from the deposited reference sequences of MCMV-bacmid pSM3fr. This prompted us to re-analyze pSM3fr and reannotate the reference sequence, as well as that for the commonly used MCMV-m157luc reporter virus. A correct reference sequence for this frequently used pSM3fr, containing a repaired version of m129 (MCK-2) and the luciferase gene instead of ORF m157, was constructed. The new reference also contains the original bacmid sequence, and it has a hybrid origin from MCMV strains Smith and K181.

Analysis of SARS-CoV-2 Spike Protein Variants with Recombinant Reporter Viruses Created from a Bacmid System.

SARS-CoV-2, the causative agent of COVID-19, has spread around the world with more than 700 million cases and 6.8 million deaths. Various variants of concern (VoC) have emerged due to mutations and recombination and concurrent selection for increased viral fitness and immune evasion. The viral protein that primarily determines the pathogenicity, infectivity, and transmissibility is the Spike protein. To analyze the specific impact of variant Spike proteins on infection dynamics, we constructed SARS-CoV-2 with a uniform B.1 backbone but with alternative Spike proteins. In addition, ORF6 was replaced by EYFP as a biological safety measure, and for use of this well-established reporter. We show that namely the delta variant Spike proteins cause a distinct phenotype from the wild type (B.1, D614G) and other variants of concern. Furthermore, we demonstrate that the omicron BA.1 Spike results in lower viral loads and a less efficient spread in vitro. Finally, we utilized viruses with the two different reporters EYFP and mCherry to establish a competitive growth assay, demonstrating that most but not all Spike variant viruses were able to outcompete wild type SARS-CoV-2 B.1.

Comparison of a Genotype 1 and a Genotype 2 Macaque Foamy Virus env Gene Indicates Distinct Infectivity and Cell-Cell Fusion but Similar Tropism and Restriction of Cell Entry by Interferon-Induced Transmembrane Proteins.

Foamy viruses (FVs) are naturally found in many different animals and also in primates with the notable exception of humans, but zoonotic infections are common. In several species, two different envelope (env) gene sequence clades or genotypes exist. We constructed a simian FV (SFV) clone containing a reporter gene cassette. In this background, we compared the env genes of the SFVmmu-DPZ9524 (genotype 1) and of the SFVmmu_R289hybAGM (genotype 2) isolates. SFVmmu_R289hybAGM env-driven infection was largely resistant to neutralization by SFVmmu-DPZ9524-neutralizing sera. While SFVmmu_R289hybAGM env consistently effected higher infectivity and cell-cell fusion, we found no differences in the cell tropism conferred by either env across a range of different cells. Infection by both viruses was weakly and non-significantly enhanced by simultaneous knockout of interferon-induced transmembrane proteins (IFITMs) 1, 2, and 3 in A549 cells, irrespective of prior interferon stimulation. Infection was modestly reduced by recombinant overexpression of IFITM3, suggesting that the SFV entry step might be weakly restricted by IFITM3 under some conditions. Overall, our results suggest that the different env gene clades in macaque foamy viruses induce genotype-specific neutralizing antibodies without exhibiting overt differences in cell tropism, but individual env genes may differ significantly with regard to fitness.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron.

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

Herpesviruses mimic zygotic genome activation to promote viral replication.

DUX4 is a germline transcription factor and a master regulator of zygotic genome activation. During early embryogenesis, DUX4 is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In adult somatic cells, DUX4 expression is silenced and its activation in adult muscle cells causes the genetic disorder Facioscapulohumeral Muscular Dystrophy (FSHD). Here we show that herpesviruses from alpha-, beta- and gamma-herpesvirus subfamilies as well as papillomaviruses actively induce DUX4 expression to promote viral transcription and replication. We demonstrate that HSV-1 immediate early proteins directly induce expression of DUX4 and its target genes including endogenous retroelements, which mimics zygotic genome activation. We further show that DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for herpesvirus infection, since genetic depletion of DUX4 by CRISPR/Cas9 abrogates viral replication. Our results show that herpesviruses induce DUX4 expression and its downstream germline-specific genes and retroelements, thus mimicking an early embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.

SARS-CoV-2 variants show temperature-dependent enhanced polymerase activity in the upper respiratory tract and high transmissibility.

With the convergent global emergence of SARS-CoV-2 variants of concern (VOC), a precise comparison study of viral fitness and transmission characteristics is necessary for the prediction of dominant VOCs and the development of suitable countermeasures. While airway temperature plays important roles in the fitness and transmissibility of respiratory tract viruses, it has not been well studied with SARS-CoV-2. Here we demonstrate that natural temperature differences between the upper (33°C) and lower (37°C) respiratory tract have profound effects on SARS-CoV-2 replication and transmission. Specifically, SARS-COV-2 variants containing the P323L or P323L/G671S mutation in the NSP12 RNA-dependent RNA polymerase (RdRp) exhibited enhanced RdRp enzymatic activity at 33°C compared to 37°C and high transmissibility in ferrets. MicroScale Thermophoresis demonstrated that the NSP12 P323L or P323L/G671S mutation stabilized the NSP12-NSP7-NSP8 complex interaction. Furthermore, reverse genetics-derived SARS-CoV-2 variants containing the NSP12 P323L or P323L/G671S mutation displayed enhanced replication at 33°C, and high transmission in ferrets. This suggests that the evolutionarily forced NSP12 P323L and P323L/G671S mutations of recent SARS-CoV-2 VOC strains are associated with increases of the RdRp complex stability and enzymatic activity, promoting the high transmissibility.

Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction.

Methylation of the mRNA 5' cap by cellular methyltransferases enables efficient translation and avoids recognition by innate immune factors. Coronaviruses encode viral 2'-O-methyltransferases to shield their RNA from host factors. Here, we generate recombinant SARS-CoV-2 harboring a catalytically inactive 2'-O-methyltransferase Nsp16, Nsp16mut, and analyze viral replication in human lung epithelial cells. Although replication is only slightly attenuated, we find SARS-CoV-2 Nsp16mut to be highly immunogenic, resulting in a strongly enhanced release of type I interferon upon infection. The elevated immunogenicity of Nsp16mut is absent in cells lacking the RNA sensor MDA5. In addition, we report that Nsp16mut is highly sensitive to type I IFN treatment and demonstrate that this strong antiviral effect of type I IFN is mediated by the restriction factor IFIT1. Together, we describe a dual role for the 2'-O-methyltransferase Nsp16 during SARS-CoV-2 replication in avoiding efficient recognition by MDA5 and in shielding its RNA from interferon-induced antiviral responses, thereby identifying Nsp16 as a promising target for generating attenuated and highly immunogenic SARS-CoV-2 strains and as a potential candidate for therapeutic intervention.

Characterization of SARS-CoV-2 Escape Mutants to a Pair of Neutralizing Antibodies Targeting the RBD and the NTD.

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.

Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases.

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

SMYD2 Inhibition Downregulates TMPRSS2 and Decreases SARS-CoV-2 Infection in Human Intestinal and Airway Epithelial Cells.

The COVID-19 pandemic caused by SARS-CoV-2 has lasted for more than two years. Despite the presence of very effective vaccines, the number of virus variants that escape neutralizing antibodies is growing. Thus, there is still a need for effective antiviral treatments that target virus replication independently of the circulating variant. Here, we show for the first time that deficiency or pharmacological inhibition of the cellular lysine-methyltransferase SMYD2 decreases TMPRSS2 expression on both mRNA and protein levels. SARS-CoV-2 uses TMPRSS2 for priming its spike protein to infect target cells. Treatment of cultured cells with the SMYD2 inhibitors AZ505 or BAY598 significantly inhibited viral replication. In contrast, treatment of Vero E6 cells, which do not express detectable amounts of TMPRSS2, had no effect on SARS-CoV-2 infection. Moreover, by generating a recombinant reporter virus that expresses the spike protein of the Delta variant of SARS-CoV-2, we demonstrate that BAY598 exhibits similar antiviral activity against this variant of concern. In summary, SMYD2 inhibition downregulates TMPRSS2 and blocks viral replication. Targeting cellular SMYD2 represents a promising tool to curtail SARS-CoV-2 infection.

Viral Mimicry of Interleukin-17A by SARS-CoV-2 ORF8.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers cytokine-mediated inflammation, leading to a myriad of clinical presentations in COVID-19. The SARS-CoV-2 open reading frame 8 (ORF8) is a secreted and rapidly evolving glycoprotein. Patients infected with SARS-CoV-2 variants with ORF8 deleted are associated with mild disease outcomes, but the molecular mechanism behind this is unknown. Here, we report that SARS-CoV-2 ORF8 is a viral cytokine that is similar to but distinct from interleukin 17A (IL-17A) as it induces stronger and broader human IL-17 receptor (hIL-17R) signaling than IL-17A. ORF8 primarily targeted blood monocytes and induced the heterodimerization of hIL-17RA and hIL-17RC, triggering a robust inflammatory response. Transcriptome analysis revealed that besides its activation of the hIL-17R pathway, ORF8 upregulated gene expression for fibrosis signaling and coagulation dysregulation. A naturally occurring ORF8 L84S variant that was highly associated with mild COVID-19 showed reduced hIL-17RA binding and attenuated inflammatory responses. This study reveals how SARS-CoV-2 ORF8 by a viral mimicry of the IL-17 cytokine contributes to COVID-19 severe inflammation. IMPORTANCE Patients infected with SARS-CoV-2 variants lacking open reading frame 8 (ORF8) have been associated with milder infection and disease outcome, but the molecular mechanism behind how this viral accessory protein mediates disease pathogenesis is not yet known. In our study, we revealed that secreted ORF8 protein mimics host IL-17 to activate IL-17 receptors A and C (IL-17RA/C) and induces a significantly stronger inflammatory response than host IL-17A, providing molecular insights into the role of ORF8 in COVID-19 pathogenesis and serving as a potential therapeutic target.