Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells.

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune-epithelial interface.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses.

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169-mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and subgenomic RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral genomic and subgenomic RNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1ß) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.

Correction: Recombinant Lloviu virus as a tool to study viral replication and host responses.

[This corrects the article DOI: 10.1371/journal.ppat.1010268.].

Recombinant Lloviu virus as a tool to study viral replication and host responses.

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

The 3' Untranslated Regions of Ebola Virus mRNAs Contain AU-Rich Elements Involved in Posttranscriptional Stabilization and Decay.

The 3' untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3' UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3' UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3' UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3' UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity.

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

Interferon-alpha or -beta facilitates SARS-CoV-2 pulmonary vascular infection by inducing ACE2.

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(ß)-two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection-but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/ß in the treatment of patients with COVID-19.

Ebolavirus polymerase uses an unconventional genome replication mechanism.

Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3' and 5' terminal nucleotides because the promoters at the 3' ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3' and 5' terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5' ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5', the 3' termini are variable and are typically 3'-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3' ends. The majority of 3' terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3' terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3'-CCUGU motif regardless of the identity of the 3' terminal nucleotide(s) and of the position of this motif relative to the 3' end. Deletion or mutation of the first residue of the 3'-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3' overhangs. This could be a mechanism to avoid antiviral recognition.

Ebolaviruses Associated with Differential Pathogenicity Induce Distinct Host Responses in Human Macrophages.

Ebola virus (EBOV) and Reston virus (RESTV) are members of the Ebolavirus genus which greatly differ in their pathogenicity. While EBOV causes a severe disease in humans characterized by a dysregulated inflammatory response and elevated cytokine and chemokine production, there are no reported disease-associated human cases of RESTV infection, suggesting that RESTV is nonpathogenic for humans. The underlying mechanisms determining the pathogenicity of different ebolavirus species are not yet known. In this study, we dissected the host response to EBOV and RESTV infection in primary human monocyte-derived macrophages (MDMs). As expected, EBOV infection led to a profound proinflammatory response, including strong induction of type I and type III interferons (IFNs). In contrast, RESTV-infected macrophages remained surprisingly silent. Early activation of IFN regulatory factor 3 (IRF3) and NF-κB was observed in EBOV-infected, but not in RESTV-infected, MDMs. In concordance with previous results, MDMs treated with inactivated EBOV and Ebola virus-like particles (VLPs) induced NF-κB activation mediated by Toll-like receptor 4 (TLR4) in a glycoprotein (GP)-dependent manner. This was not the case in cells exposed to live RESTV, inactivated RESTV, or VLPs containing RESTV GP, indicating that RESTV GP does not trigger TLR4 signaling. Our results suggest that the lack of immune activation in RESTV-infected MDMs contributes to lower pathogenicity by preventing the cytokine storm observed in EBOV infection. We further demonstrate that inhibition of TLR4 signaling abolishes EBOV GP-mediated NF-κB activation. This finding indicates that limiting the excessive TLR4-mediated proinflammatory response in EBOV infection should be considered as a potential supportive treatment option for EBOV disease.IMPORTANCE Emerging infectious diseases are a major public health concern, as exemplified by the recent devastating Ebola virus (EBOV) outbreak. Different ebolavirus species are associated with widely varying pathogenicity in humans, ranging from asymptomatic infections for Reston virus (RESTV) to severe disease with fatal outcomes for EBOV. In this comparative study of EBOV- and RESTV-infected human macrophages, we identified key differences in host cell responses. Consistent with previous data, EBOV infection is associated with a proinflammatory signature triggered by the surface glycoprotein (GP), which can be inhibited by blocking TLR4 signaling. In contrast, infection with RESTV failed to stimulate a strong host response in infected macrophages due to the inability of RESTV GP to stimulate TLR4. We propose that disparate proinflammatory host signatures contribute to the differences in pathogenicity reported for ebolavirus species and suggest that proinflammatory pathways represent an intriguing target for the development of novel therapeutics.

Filovirus Strategies to Escape Antiviral Responses.

This chapter describes the various strategies filoviruses use to escape host immune responses with a focus on innate immune and cell death pathways. Since filovirus replication can be efficiently blocked by interferon (IFN), filoviruses have evolved mechanisms to counteract both type I IFN induction and IFN response signaling pathways. Intriguingly, marburg- and ebolaviruses use different strategies to inhibit IFN signaling. This chapter also summarizes what is known about the role of IFN-stimulated genes (ISGs) in filovirus infection. These fall into three categories: those that restrict filovirus replication, those whose activation is inhibited by filoviruses, and those that have no measurable effect on viral replication. In addition to innate immunity, mammalian cells have evolved strategies to counter viral infections, including the induction of cell death and stress response pathways, and we summarize our current knowledge of how filoviruses interact with these pathways. Finally, this chapter delves into the interaction of EBOV with myeloid dendritic cells and macrophages and the associated inflammatory response, which differs dramatically between these cell types when they are infected with EBOV. In summary, we highlight the multifaceted nature of the host-viral interactions during filoviral infections.

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions.

UNLABELLED: A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. IMPORTANCE: Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families.

UNLABELLED: Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5'-triphosphates (5'-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5'-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1(-/-) and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1(-/-) and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5' ends of IAV gene segments. The affinity for 5'-ppp RNA was approximately 10-fold lower than that for non-2'-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. IMPORTANCE: Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.

Analysis of the highly diverse gene borders in Ebola virus reveals a distinct mechanism of transcriptional regulation.

UNLABELLED: Ebola virus (EBOV) belongs to the group of nonsegmented negative-sense RNA viruses. The seven EBOV genes are separated by variable gene borders, including short (4- or 5-nucleotide) intergenic regions (IRs), a single long (144-nucleotide) IR, and gene overlaps, where the neighboring gene end and start signals share five conserved nucleotides. The unique structure of the gene overlaps and the presence of a single long IR are conserved among all filoviruses. Here, we sought to determine the impact of the EBOV gene borders during viral transcription. We show that readthrough mRNA synthesis occurs in EBOV-infected cells irrespective of the structure of the gene border, indicating that the gene overlaps do not promote recognition of the gene end signal. However, two consecutive gene end signals at the VP24 gene might improve termination at the VP24-L gene border, ensuring efficient L gene expression. We further demonstrate that the long IR is not essential for but regulates transcription reinitiation in a length-dependent but sequence-independent manner. Mutational analysis of bicistronic minigenomes and recombinant EBOVs showed no direct correlation between IR length and reinitiation rates but demonstrated that specific IR lengths not found naturally in filoviruses profoundly inhibit downstream gene expression. Intriguingly, although truncation of the 144-nucleotide-long IR to 5 nucleotides did not substantially affect EBOV transcription, it led to a significant reduction of viral growth. IMPORTANCE: Our current understanding of EBOV transcription regulation is limited due to the requirement for high-containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative-sense RNA viruses. A single polymerase entry site at the 3' end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from that in prototype viruses and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle.

Ebola virus does not block apoptotic signaling pathways.

Since viruses rely on functional cellular machinery for efficient propagation, apoptosis is an important mechanism to fight viral infections. In this study, we sought to determine the mechanism of cell death caused by Ebola virus (EBOV) infection by assaying for multiple stages of apoptosis and hallmarks of necrosis. Our data indicate that EBOV does not induce apoptosis in infected cells but rather leads to a nonapoptotic form of cell death. Ultrastructural analysis confirmed necrotic cell death of EBOV-infected cells. To investigate if EBOV blocks the induction of apoptosis, infected cells were treated with different apoptosis-inducing agents. Surprisingly, EBOV-infected cells remained sensitive to apoptosis induced by external stimuli. Neither receptor- nor mitochondrion-mediated apoptosis signaling was inhibited in EBOV infection. Although double-stranded RNA (dsRNA)-induced activation of protein kinase R (PKR) was blocked in EBOV-infected cells, induction of apoptosis mediated by dsRNA was not suppressed. When EBOV-infected cells were treated with dsRNA-dependent caspase recruiter (dsCARE), an antiviral protein that selectively induces apoptosis in cells containing dsRNA, virus titers were strongly reduced. These data show that the inability of EBOV to block apoptotic pathways may open up new strategies toward the development of antiviral therapeutics.

Modulations of Homeostatic ACE2, CD147, GRP78 Pathways Correlate with Vascular and Endothelial Performance Markers during Pulmonary SARS-CoV-2 Infection.

The pathologic consequences of Coronavirus Disease-2019 (COVID-19) include elevated inflammation and dysregulated vascular functions associated with thrombosis. In general, disruption of vascular homeostasis and ensuing prothrombotic events are driven by activated platelets, monocytes, and macrophages, which form aggregates (thrombi) attached to the endothelium lining of vessel walls. However, molecular pathways underpinning the pathological interactions between myeloid cells and endothelium during COVID-19 remain undefined. Here, we tested the hypothesis that modulations in the expression of cellular receptors angiotensin-converting enzyme 2 (ACE2), CD147, and glucose-regulated protein 78 (GRP78), which are involved in homeostasis and endothelial performance, are the hallmark responses induced by SARS-CoV-2 infection. Cultured macrophages and lungs of hamster model systems were used to test this hypothesis. The results indicate that while macrophages and endothelial cells are less likely to support SARS-CoV-2 proliferation, these cells may readily respond to inflammatory stimuli generated by the infected lung epithelium. SARS-CoV-2 induced modulations of tested cellular receptors correlated with corresponding changes in the mRNA expression of coagulation cascade regulators and endothelial integrity components in infected hamster lungs. Among these markers, tissue factor (TF) had the best correlation for prothrombotic events during SARS-CoV-2 infection. Furthermore, the single-molecule fluorescence in situ hybridization (smFISH) method alone was sufficient to determine the peak and resolution phases of SARS-CoV-2 infection and enabled screening for cellular markers co-expressed with the virus. These findings suggest possible molecular pathways for exploration of novel drugs capable of blocking the prothrombotic shift events that exacerbate COVID-19 pathophysiology and control the disease.

Minor intron-containing genes as an ancient backbone for viral infection?

Minor intron-containing genes (MIGs) account for <2% of all human protein-coding genes and are uniquely dependent on the minor spliceosome for proper excision. Despite their low numbers, we surprisingly found a significant enrichment of MIG-encoded proteins (MIG-Ps) in protein-protein interactomes and host factors of positive-sense RNA viruses, including SARS-CoV-1, SARS-CoV-2, MERS coronavirus, and Zika virus. Similarly, we observed a significant enrichment of MIG-Ps in the interactomes and sets of host factors of negative-sense RNA viruses such as Ebola virus, influenza A virus, and the retrovirus HIV-1. We also found an enrichment of MIG-Ps in double-stranded DNA viruses such as Epstein-Barr virus, human papillomavirus, and herpes simplex viruses. In general, MIG-Ps were highly connected and placed in central positions in a network of human-host protein interactions. Moreover, MIG-Ps that interact with viral proteins were enriched with essential genes. We also provide evidence that viral proteins interact with ancestral MIGs that date back to unicellular organisms and are mainly involved in basic cellular functions such as cell cycle, cell division, and signal transduction. Our results suggest that MIG-Ps form a stable, evolutionarily conserved backbone that viruses putatively tap to invade and propagate in human host cells.

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers.

Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results.