Tetherin restricts SARS-CoV-2 replication despite antagonistic effects of Spike and ORF7a (preprint)

SARS-CoV-2 infection induces interferon-stimulated genes, one of which encodes Tetherin, a transmembrane protein inhibiting the release of various enveloped viruses from infected cells. Previous studies revealed that SARS-CoV encodes two Tetherin antagonists: the Spike protein (S) inducing lysosomal degradation of Tetherin, and ORF7a altering its glycosylation. SARS-CoV-2 ORF7a has also been shown to antagonize Tetherin. Therefore, we here investigated whether SARS-CoV-2 S is also a Tetherin antagonist and compared the abilities and mechanisms of S and ORF7a in counteracting Tetherin. SARS-CoV and SARS-CoV-2 S reduced Tetherin cell surface levels in a cell type-dependent manner, possibly related to the basal protein levels of Tetherin. In HEK293T cells, under conditions of high exogenous Tetherin expression, SARS-CoV-2 S and ORF7a reduced total Tetherin levels much more efficiently than the respective counterparts derived from SARS-CoV. Nevertheless, ORF7a from both strains was able to alter Tetherin glycosylation. The ability to decrease total protein levels of Tetherin was conserved among S proteins from different SARS-CoV-2 variants (D614G, Cluster 5, , {gamma}, {delta}, o). While SARS-CoV-2 S and ORF7a both colocalized with Tetherin, only ORF7a directly interacted with the restriction factor. Despite the presence of two Tetherin antagonists, however, SARS-CoV-2 replication in Caco-2 cells was further enhanced upon Tetherin knockout. Altogether, our data show that endogenous Tetherin restricts SARS-CoV-2 replication, and that the antiviral activity of Tetherin is partially counteracted by two viral antagonists with differential and complementary modes of action, S and ORF7a.

SARS-CoV-2 infection activates endogenous retroviruses of the LTR69 subfamily (preprint)

Accumulating evidence suggests that endogenous retroviruses (ERVs) play an important role in the host response to infection and the development of disease. By combining RNA- and ChIP-sequencing analyses with RT-qPCR, we show that SARS-CoV-2 infection induces the LTR69 subfamily of ERVs, both in vitro and in vivo. Using functional assays, we identified one SARS-CoV-2-activated LTR69 locus, termed Dup69, which exhibits enhancer activity and is responsive to the transcription factors IRF3 and p65/RelA. LTR69-Dup69 is located about 500 bp upstream of a long non-coding RNA gene (ENSG00000289418) and within the PTPRN2 gene encoding a diabetes-associated autoantigen. Both ENSG00000289418 and PTPRN2 showed a significant increase in expression upon SARS-CoV-2 infection. Thus, our study sheds light on the interplay of exogenous with endogenous viruses and helps to understand how ERVs regulate gene expression during infection.

SARS-CoV-2 ORF3c suppresses immune activation by inhibiting innate sensing (preprint)

SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While most of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. For example, the ORF3a sgRNA also encodes ORF3c, an enigmatic 41-amino acid peptide. Here, we show that ORF3c suppresses RIG-I- and MDA5-mediated immune activation and interacts with the signaling adaptor MAVS. In line with this, ORF3c inhibits IFN-{beta} induction. This 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. In agreement with this, disruption of ORF3c did not significantly affect SARS-CoV-2 replication in CaCo-2 or CaLu-3 cells. In summary, we here identify ORF3c as an immune evasion factor that suppresses IFN-{beta} induction, but is dispensable for efficient replication of SARS-CoV-2.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition (preprint)

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

A Common Methodological Phylogenomics Framework for intra-patient heteroplasmies to infer SARS-CoV-2 sublineages and tumor clones (preprint)

We present a common methodological framework to infer the phylogenomics from genomic data, be it reads of SARS-CoV-2 of multiple COVID-19 patients or bulk DNAseq of the tumor of a cancer patient. The commonality is in the phylogenetic retrodiction based on the genomic reads in both scenarios. While there is evidence of heteroplasmy, i.e., multiple lineages of SARS-CoV-2 in the same COVID-19 patient; to date, there is no evidence of sublineages recombining within the same patient. The heterogeneity in a patient's tumor is analogous to intra-patient heteroplasmy and the absence of recombination in the cells of tumor is a widely accepted assumption. Just as the different frequencies of the genomic variants in a tumor presupposes the existence of multiple tumor clones and provides a handle to computationally infer them, we postulate that so do the different variant frequencies in the viral reads, offering the means to infer the multiple co-infecting sublineages. We describe the Concerti computational framework for inferring phylogenies in each of the two scenarios. To demonstrate the accuracy of the method, we reproduce some known results in both scenarios. We also make some additional discoveries. We uncovered new potential parallel mutation in the evolution of the SARS-CoV-2 virus. In the context of cancer, we uncovered new clones harboring resistant mutations to therapy from clinically plausible phylogenetic tree in a patient.

Systematic analysis of innate immune antagonism reveals vulnerabilities of SARS-CoV-2 (preprint)

The innate immune system constitutes a powerful barrier against viral infections. However, it may fail because successful emerging pathogens, like SARS-CoV-2, evolved strategies to counteract it. Here, we systematically assessed the impact of 29 SARS-CoV-2 proteins on viral sensing, type I, II and III interferon (IFN) signaling, autophagy and inflammasome formation. Mechanistic analyses show that autophagy and type I IFN responses are effectively counteracted at different levels. For example, Nsp14 induces loss of the IFN receptor, whereas ORF3a disturbs autophagy at the Golgi/endosome interface. Comparative analyses revealed that antagonism of type I IFN and autophagy is largely conserved, except that SARS-CoV-1 Nsp15 is more potent in counteracting type I IFN than its SARS-CoV-2 ortholog. Altogether, however, SARS-CoV-2 counteracts type I IFN responses and autophagy much more efficiently than type II and III IFN signaling. Consequently, the virus is relatively resistant against exogenous IFN-/{beta} and autophagy modulation but remains highly vulnerable towards IFN-{gamma} and -{lambda} treatment. In combination, IFN-{gamma} and -{lambda} act synergistically, and drastically reduce SARS-CoV-2 replication at exceedingly low doses. Our results identify ineffective type I and II antagonism as weakness of SARS-CoV-2 that may allow to devise safe and effective anti-viral therapies based on targeted innate immune activation.

Integrated characterization of SARS-CoV-2 genome, microbiome, antibiotic resistance and host response from single throat swabs (preprint)

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a newly-developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.

Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without (preprint)

Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split {beta}-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.

Sarbecovirus ORF6 Proteins Hamper the Induction of Interferon Signaling by Blocking mRNA Nuclear Export (preprint)

The presence of an ORF6 gene distinguishes Sarbecoviruses such as SARS-CoV and SARS-CoV-2 from other Betacoronaviruses. Here, we show that ORF6 inhibits the induction of type I IFN upon viral infection, as well as IFN types I and III signaling. Intriguingly, the anti-IFN activity of ORF6 proteins of SARS-CoV-2 lineages is more potent than that of SARS-CoV lineages. Mutational analyses identified residues E46 and Q56 as determinants of the potent IFN-antagonistic activity of SARS-CoV-2 ORF6. Moreover, we show that ORF6 binds to RAE1 and NUP98 via its C-terminus, thereby inhibiting the nuclear export of IFNB1 mRNA. Finally, we identify natural occurring frameshift/nonsense mutations that result in an inactivating truncation of ORF6 in approximately 0.2% of SARS-CoV-2 isolates. Altogether, our findings suggest that ORF6 contributes to the poor IFN activation observed in COVID-19 patients. Furthermore, the emergence of SARS-CoV-2 variants without functional ORF6 may contribute to the attenuation of viral pathogenicity.Funding: This study was supported in part by AMED Research Program on Emerging and Re-emerging Infectious Diseases 20fk0108146 (to K.S.), 19fk0108171 (to S.N. and K.S.) and 20fk0108270 (to K.S.); AMED Research Program on HIV/AIDS 19fk0410019 (to K.S.) and 20fk0410014 (to K.S.); JST J-RAPID JPMJJR2007 (to K.S.); KAKENHI Grant-in-Aid for Scientific Research B 18H02662 (to K.S.), KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas 16H06429 (to S.N. and K.S.), 16K21723 (to S.N. and K.S.), 17H05823 (to S.N.), 17H05813 (to K.S.), 19H04843 (to S.N.) and 19H04826 (to K.S.), and Fund for the Promotion of Joint International Research (Fostering Joint International Research) 18KK0447 (to K.S.); JSPS Research Fellow DC1 19J20488 (to I.K.) and DC1 19J22914 (to Y.K.); ONO Medical Research Foundation (to K.S.); Ichiro Kanehara Foundation (to K.S.); Lotte Foundation (to K.S.); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to K.S.); Daiichi Sankyo Foundation of Life Science (to K.S.); Sumitomo Foundation (to K.S.); Uehara Foundation (to K.S.); Takeda Science Foundation (to K.S.); JSPS Core-to-Core program (A. Advanced Research Networks) (to K.S.); the Canon Foundation in Europe (to. D.S. and K.S.); a COVID19 Research Grant of the Federal Ministry of Education and Research (MWK) Baden-Württemberg (to. D.S.); 2020 Tokai University School of Medicine Research Aid (to S.N.); and International Joint Research Project of the Institute of Medical Science, the University of Tokyo 2020-K3003 (to D.S. and K.S.). Conflict of Interest: The authors declare that no competing interests exist.

IFITM proteins promote SARS-CoV-2 infection in human lung cells (preprint)

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) restrict numerous viral pathogens and are thought to prevent infection by severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudoparticle infection of cells artificially overexpressing IFITMs. Here, we confirmed that overexpression of IFITMs blocks pseudoparticle infections mediated by the Spike proteins of {beta}-coronaviruses including pandemic SARS-CoV-2. In striking contrast, however, endogenous IFITM expression promoted genuine SARS-CoV-2 infection in human lung cells both in the presence and absence of interferon. IFITM2 was most critical for efficient entry of SARS-CoV-2 and enhanced virus production from Calu-3 cells by several orders of magnitude. IFITMs are expressed and further induced by interferons in the lung representing the primary site of SARS-CoV-2 infection as well as in other relevant tissues. Our finding that IFITMs enhance SARS-CoV-2 infection under conditions approximating the in vivo situation shows that they may promote viral invasion during COVID-19. HIGHLIGHTSO_LIOverexpression of IFITM1, 2 and 3 restricts SARS-CoV-2 infection C_LIO_LIEndogenous IFITM1, 2 and 3 boost SARS-CoV-2 infection of human lung cells C_LIO_LIIFITM2 is critical for efficient entry of SARS-CoV-2 in Calu-3 cells C_LI

Seeding of outbreaks of COVID-19 by contaminated fresh and frozen food (preprint)

An explanation is required for the re-emergence of COVID-19 outbreaks in regions with apparent local eradication. Recent outbreaks have emerged in Vietnam, New Zealand and parts of China where there had been no cases for some months. Importation of contaminated food and food packaging is a feasible source for such outbreaks and a source of clusters within existing outbreaks. Such events can be prevented if the risk is better appreciated.

SARS-CoV-2 Infection and Transmission Depends on Heparan Sulfates and Is Blocked by Low Molecular Weight Heparins (preprint)

The current pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and new outbreaks worldwide highlight the need for preventive treatments. Although angiotensin converting enzyme 2 (ACE2) is the primary receptor for SARS-CoV-2, we identified heparan sulfate proteoglycans expressed by epithelial cells, alveolar macrophages and dendritic cells as co-receptors for SARS-CoV-2. Low molecular weight heparins (LMWH) blocked SARS-CoV-2 infection of epithelial cells and alveolar macrophages, and virus dissemination by dendritic cells. Notably, potent neutralizing antibodies from COVID-19 patients interfered with SARS-CoV-2 binding to heparan sulfate proteoglycans, underscoring the importance of heparan sulfate proteoglycans as receptors and uncover that SARS-CoV-2 binding to heparan sulfates is an important mechanism for neutralization. These results have imperative implications for our understanding of SARS-CoV-2 host cell entry and reveal an important target for novel prophylactic intervention.

The Zinc Finger Antiviral Protein restricts SARS-CoV-2 (preprint)

Recent evidence shows that the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly sensitive to interferons (IFNs). However, the underlying antiviral effectors remain to be defined. Here, we show that Zinc finger antiviral protein (ZAP) that specifically targets CpG dinucleotides in viral RNA sequences restricts SARS-CoV-2. We demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Strikingly, SARS-CoV-2 and its closest relatives from bats show the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, knock-down of ZAP significantly increased SARS-CoV-2 production in lung cells, particularly upon treatment with IFN- or IFN-{gamma}. Thus, our results identify ZAP as an effector of the IFN response against SARS-CoV-2, although this pandemic pathogen may be preadapted to the low CpG environment in humans. HighlightsO_LISARS-CoV-2 and its closest bat relatives show strong CpG suppression C_LIO_LIIFN-{beta}, -{gamma} and -{lambda} inhibit SARS-CoV-2 with high efficiency C_LIO_LIZAP restricts SARS-CoV-2 and contributes to the antiviral effect of IFNs C_LI