SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

Spread and clinical severity of respiratory syncytial virus A genotype ON1 in Germany, 2011-2017.

BACKGROUND: The Respiratory Syncytial Virus (RSV) A genotype ON1, which was first detected in Ontario (Canada) in 2010/11, appeared in Germany in 2011/12. Preliminary observations suggested a higher clinical severity in children infected with this new genotype. We investigated spread and disease severity of RSV-A ON1 in pediatric in- and outpatient settings. METHODS: During 2010/11 to 2016/17, clinical characteristics and respiratory samples from children with acute respiratory tract infections (RTI) were obtained from ongoing surveillance studies in 33 pediatric practices (PP), one pediatric hospital ward (PW) and 23 pediatric intensive care units (PICU) in Germany. RSV was detected in the respiratory samples by PCR; genotypes were identified by sequencing. Within each setting, clinical severity markers were compared between RSV-A ON1 and RSV-A non-ON1 genotypes. RESULTS: A total of 603 children with RSV-RTI were included (132 children in PP, 288 in PW, and 183 in PICU). Of these children, 341 (56.6%) were infected with RSV-A, 235 (39.0%) with RSV-B, and one child (0.2%) with both RSV-A and RSV-B; in 26 (4.3%) children, the subtype could not be identified. In the 341 RSV-A positive samples, genotype ON1 was detected in 247 (72.4%), NA1 in 92 (26.9%), and GA5 in 2 children (0.6%). RSV-A ON1, rarely observed in 2011/12, was the predominant RSV-A genotype in all settings by 2012/13 and remained predominant until 2016/17. Children in PP or PW infected with RSV-A ON1 did not show a more severe clinical course of disease compared with RSV-A non-ON1 infections. In the PICU group, hospital stay was one day longer (median 8 days, inter-quartile range (IQR) 7-12 vs. 7 days, IQR 5-9; p = 0.02) and duration of oxygen treatment two days longer (median 6 days, IQR 4-9 vs. 4 days, IQR 2-6; p = 0.03) for children infected with RSV-A ON1. CONCLUSIONS: In children, RSV-A ON1 largely replaced RSV-A non-ON1 genotypes within two seasons and remained the predominant RSV-A genotype in Germany during subsequent seasons. A higher clinical severity of RSV-A ON1 was observed within the group of children receiving PICU treatment, whereas in other settings clinical severity of RSV-A ON1 and non-ON1 genotypes was largely similar.

In situ evolution of virus-specific cytotoxic T cell responses in the lung.

Cytotoxic T cells (CTL) play a critical role in the clearance of respiratory viral infections, but they also contribute to disease manifestations. In this study, we infected mice with a genetically modified pneumonia virus of mice (PVM) that allowed visualization of virus-specific CTL and infected cells in situ. The first virus-specific T cells entered the lung via blood vessels in the scattered foci of PVM-infected cells, which densely clustered around the bronchi at day 7 after infection. At this time, overall pulmonary virus load was maximal, but the mice showed no overt signs of disease. On days 8 to 9, T cells gained access to the infected bronchial epithelium and to the lung interstitium, which was associated with a reduction in the number of virus-infected cells within the initial clusters but could not prevent further virus spread throughout the lung tissue. Interestingly, recruitment of virus-specific CTL throughout the parenchyma was still ongoing on day 10, when the virus infection was already largely controlled. This also represented the peak of clinical disease. Thus, disease was associated with an exuberant T cell infiltration late in the course of the infection, which may be required to completely eliminate virus at residual foci of infection. PVM-induced immunopathology may thus result from the need to generate widespread T cell infiltrates to complete the elimination of virus-infected cells in a large organ like the lung. This experimental model provides the first insights into the spatiotemporal evolution of pulmonary antiviral T cell immunity in vivo.

SARS-CoV-2 tropism to intestinal but not gastric epithelial cells is defined by limited ACE2 expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primarily affects the lung but can also cause gastrointestinal (GI) symptoms. In vitro experiments confirmed that SARS-CoV-2 robustly infects intestinal epithelium. However, data on infection of adult gastric epithelium are sparse and a side-by-side comparison of the infection in the major segments of the GI tract is lacking. We provide this direct comparison in organoid-derived monolayers and demonstrate that SARS-CoV-2 robustly infects intestinal epithelium, while gastric epithelium is resistant to infection. RNA sequencing and proteome analysis pointed to angiotensin-converting enzyme 2 (ACE2) as a critical factor, and, indeed, ectopic expression of ACE2 increased susceptibility of gastric organoid-derived monolayers to SARS-CoV-2. ACE2 expression pattern in GI biopsies of patients mirrors SARS-CoV-2 infection levels in monolayers. Thus, local ACE2 expression limits SARS-CoV-2 expression in the GI tract to the intestine, suggesting that the intestine, but not the stomach, is likely to be important in viral replication and possibly transmission.

Evaluation of pneumonia virus of mice as a possible human pathogen.

Pneumonia virus of mice (PVM), a relative of human respiratory syncytial virus (RSV), causes respiratory disease in mice. There is serologic evidence suggesting widespread exposure of humans to PVM. To investigate replication in primates, African green monkeys (AGM) and rhesus macaques (n = 4) were inoculated with PVM by the respiratory route. Virus was shed intermittently at low levels by a subset of animals, suggesting poor permissiveness. PVM efficiently replicated in cultured human cells and inhibited the type I interferon (IFN) response in these cells. This suggests that poor replication in nonhuman primates was not due to a general nonpermissiveness of primate cells or poor control of the IFN response. Seroprevalence in humans was examined by screening sera from 30 adults and 17 young children for PVM-neutralizing activity. Sera from a single child (6%) and 40% of adults had low neutralizing activity against PVM, which could be consistent with increasing incidence of exposure following early childhood. There was no cross-reaction of human or AGM sera between RSV and PVM and no cross-protection in the mouse model. In native Western blots, human sera reacted with RSV but not PVM proteins under conditions in which AGM immune sera reacted strongly. Serum reactivity was further evaluated by flow cytometry using unfixed Vero cells infected with PVM or RSV expressing green fluorescent protein (GFP) as a measure of viral gene expression. The reactivity of human sera against RSV-infected cells correlated with GFP expression, whereas reactivity against PVM-infected cells was low and uncorrelated with GFP expression. Thus, PVM specificity was not evident. Our results indicate that the PVM-neutralizing activity of human sera is not due to RSV- or PVM-specific antibodies but may be due to low-affinity, polyreactive natural antibodies of the IgG subclass. The absence of PVM-specific antibodies and restriction in nonhuman primates makes PVM unlikely to be a human pathogen.

Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection.

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vß8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vß9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vß8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.

Both nonstructural proteins NS1 and NS2 of pneumonia virus of mice are inhibitors of the interferon type I and type III responses in vivo.

Infection of mice with pneumonia virus of mice (PVM) provides a convenient experimental pathogenesis model in a natural host for a human respiratory syncytial virus-related virus. Extending our previous work showing that the PVM nonstructural (NS) proteins were pathogenicity factors in mice, we identify both the NS1 and NS2 proteins as antagonists of alpha/beta interferon (IFN-α/ß) and IFN-λ by use of recombinant PVM (rPVM) with single and combined deletions of the NS proteins (ΔNS1, ΔNS2, and ΔNS1 ΔNS2). Wild-type and NS deletion PVMs were evaluated for growth and pathogenesis by infecting knockout mice that lack functional receptors to IFN-α/ß, IFN-λ, or both. The absence of the receptor to IFN-α/ß (IFNAR) or IFN-λ (interleukin-28 receptor α chain [IL-28Rα]) individually did not reverse the attenuated virulence of the NS deletion viruses although loss of IFNAR partially restored replication efficiency. When both receptors were deleted, replication and virulence were largely rescued for rPVM ΔNS1 and were significantly but not completely rescued for rPVM ΔNS2. As for rPVM ΔNS1 ΔNS2, the effect was mostly limited to partial enhancement of replication. This indicates that both IFN-α/ß and IFN-λ contributed to restricting the NS deletion viruses, with the former playing the greater role. Interestingly, the replication and virulence of wild-type PVM were completely unaffected by the presence or absence of functional receptors to IFN-α/ß and IFN-λ, indicating that both systems are strongly suppressed during infection. However, pretreatment of mice with IFN-α/ß was protective against lethal rPVM challenge, whereas pretreatment with IFN-λ delayed but did not prevent disease and, in some cases, reduced mortality. The fact that virulence of rPVM lacking NS2 was not recovered completely when both interferon receptors were deleted suggests that NS2 may have further functions outside the IFN system.

Deletion of nonstructural proteins NS1 and NS2 from pneumonia virus of mice attenuates viral replication and reduces pulmonary cytokine expression and disease.

Pneumonia virus of mice (PVM) strain 15 causes fatal pneumonia in mice and provides a convenient model for human respiratory syncytial virus pathogenesis and immunobiology. We prepared PVM mutants lacking the genes for nonstructural proteins NS1 and/or NS2. In Vero cells, which lack type I interferon (IFN), deletion of these proteins had no effect on the efficiency of virus growth. In IFN-competent mouse embryo fibroblasts, wild-type (wt) PVM and the DeltaNS1 virus grew efficiently and strongly inhibited the IFN response, whereas virus lacking NS2 was highly attenuated and induced high levels of IFN and IFN-inducible genes. In BALB/c mice, intranasal infection with wt PVM caused overt disease that began on day 6 and was lethal by day 9 postinoculation. In comparison, DeltaNS1 induced transient, reduced disease, and DeltaNS2 and DeltaNS12 caused no disease. Thus, NS1 and NS2 are virulence factors, with NS2 being a major antagonist of the type I IFN system. The pulmonary titers of wt PVM and DeltaNS1 were high on day 3 and increased further by day 6; in addition, expression of IFN and representative proinflammatory cytokines/chemokines and T lymphocyte-related cytokines was undetectable on day 3 but increased dramatically by day 6 coincident with the onset of disease. The titers of DeltaNS2 and DeltaNS12 were somewhat lower on day 3 and decreased further by day 6; in addition, these viruses induced a more circumscribed set of cytokines/chemokines (IFN, interleukin-6 [IL-6], and CXCL10) that were detected on day 3 and had largely subsided by day 6. Lung immunohistology revealed abundant PVM-positive pneumocytes and bronchial and bronchiolar epithelial cells in wt PVM- and DeltaNS1-infected mice on day 6 compared to few PVM-positive foci with DeltaNS2 and DeltaNS12. These results indicate that severe PVM disease is associated with high, poorly controlled virus replication driving the expression of high levels of pulmonary IFN and a broad array of cytokines/chemokines. In contrast, in the absence of NS2, there was an early, transient innate response involving moderate levels of IFN, IL-6, and CXCL10 that restricted virus replication and prevented disease.

Role of T cells in virus control and disease after infection with pneumonia virus of mice.

Infection of mice with pneumonia virus of mice (PVM) is used as a natural host experimental model for studying the pathogenesis of infection with the closely related human respiratory syncytial virus. We analyzed the contribution of T cells to virus control and pathology after PVM infection. Control of a sublethal infection with PVM strain 15 in C57BL/6 mice was accompanied by a 100-fold increase in pulmonary cytotoxic T lymphocytes, 20% of which were specific for PVM. T-cell-deficient mice failed to eliminate PVM and became virus carriers in the absence of the clinical or histopathological signs of pneumonia that occurred after infection of control mice. Mice with limited T-cell numbers did not achieve virus control without weight loss, indicating that T-cell-mediated virus control was closely linked to immunopathology. Both CD4 and CD8 T cells independently contributed to virus elimination and disease. Virus control and disease were similar in the absence of perforin, gamma interferon, or tumor necrosis factor alpha. Interestingly, disease and mortality after lethal high-dose PVM infection were independent of T cells. These data illustrate a key role for T cells in control of PVM infection and demonstrate that both T-cell-dependent and -independent pathways contribute to disease in a viral dose-dependent fashion.

Increase in CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase in CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis.

S. mansoni bolsters anti-viral immunity in the murine respiratory tract.

The human intestinal parasite Schistosoma mansoni causes a chronic disease, schistosomiasis or bilharzia. According to the current literature, the parasite induces vigorous immune responses that are controlled by Th2 helper cells at the expense of Th1 helper cells. The latter cell type is, however, indispensable for anti-viral immune responses. Remarkably, there is no reliable literature among 230 million patients worldwide describing defective anti-viral immune responses in the upper respiratory tract, for instance against influenza A virus or against respiratory syncitial virus (RSV). We therefore re-examined the immune response to a human isolate of S. mansoni and challenged mice in the chronic phase of schistosomiasis with influenza A virus, or with pneumonia virus of mice (PVM), a mouse virus to model RSV infections. We found that mice with chronic schistosomiasis had significant, systemic immune responses induced by Th1, Th2, and Th17 helper cells. High serum levels of TNF-α, IFN-γ, IL-5, IL-13, IL-2, IL-17, and GM-CSF were found after mating and oviposition. The lungs of diseased mice showed low-grade inflammation, with goblet cell hyperplasia and excessive mucus secretion, which was alleviated by treatment with an anti-TNF-α agent (Etanercept). Mice with chronic schistosomiasis were to a relative, but significant extent protected from a secondary viral respiratory challenge. The protection correlated with the onset of oviposition and TNF-α-mediated goblet cell hyperplasia and mucus secretion, suggesting that these mechanisms are involved in enhanced immune protection to respiratory viruses during chronic murine schistosomiasis. Indeed, also in a model of allergic airway inflammation mice were protected from a viral respiratory challenge with PVM.

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R.

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

Influence of a single viral epitope on T cell response and disease after infection of mice with respiratory syncytial virus.

CTL are important for virus clearance but also contribute to immunopathology after the infection of BALB/c mice with respiratory syncytial virus (RSV). The pulmonary immune response to RSV is dominated by a CTL population directed against the CTL epitope M2-1 82-90. Infection with a virus carrying an M2-1 N89A mutation introduced by reverse genetics failed to activate this immunodominant CTL population, leading to a significant decrease in the overall antiviral CTL response. There was no compensatory increase in responses to the mutated epitope, to the subdominant epitope F 85-93, or to yet undefined minor epitopes in the N or the P protein. However, there was some increase in the response to the subdominant epitope M2-1 127-135, which is located in the same protein and presented by the same H-2Kd MHC molecule. Infection with the mutant virus reversed the oligoclonality of the T cell response elicited by the wild-type virus. These changes in the pattern and composition of the antiviral CTL response only slightly impaired virus clearance but significantly reduced RSV-induced weight loss. These data illustrate how T cell epitope mutations can influence the virus-host relationship and determine disease after an acute respiratory virus infection.

Identification of a novel virulence factor in recombinant pneumonia virus of mice.

Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (Delta G) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the Delta G mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

Complete sequence of the RNA genome of pneumonia virus of mice (PVM).

Pneumonia virus of mice (PVM) is an enveloped RNA-containing virus of Family Paramyxoviridae. Sequences had been determined previously for a number of PVM genes, although these represented cloned cDNAs rather than consensus sequences. Sequences were not available for the 3' -leader and 5' -trailer regions that constitute the genome termini or for the large polymerase L gene that accounts for 43% of the genome. Also, the available sequences were from an attenuated variant of strain 15, whereas the present study analyzed the version of strain 15 that is available from the American Type Culture Collection (ATCC) and is highly virulent in mice. Analysis of unclosed RT-PCR products yielded a complete consensus sequence of 14,886 nt (GenBank accession number AY729016). Of the regions for which sequences had been previously reported for the non-pathogenic strain, there were 13 nucleotide differences and 10 amino acid differences compared to the present consensus sequence for the virulent isolate. The various genes of PVM shared 29-62% nucleotide sequence identity and 10-60% amino acid sequence identity with human or bovine respiratory syncytial virus (HRSV and BRSV), its closest relatives.

Reevaluation of the virulence of prototypic strain 15 of pneumonia virus of mice.

Prototypic strain 15 of pneumonia virus of mice (PVM) has been described as being nonpathogenic in mice, in contrast to the mouse-passaged, highly virulent strain J3666. Previous sequence analysis also indicated that strain 15 encodes an attachment G protein that is truncated at the amino terminus, which for the amino terminally anchored protein deletes the cytoplasmic tail. However, we found that PVM strain 15 obtained from the American Type Culture Collection was highly virulent in mice and was essentially indistinguishable on that basis from strain J3666. Sequence analysis showed that this preparation of virus encodes a G protein with an intact cytoplasmic tail: the truncated predicted protein in the previous sequence appeared to be due to a single nucleotide insertion that disrupted the upstream end of the open reading frame and shifted the translational start site to the next downstream AUG. Taken together, the two studies indicate that strain 15 is an inherently virulent strain but that a nonpathogenic variant that was generated during passage in vitro and encodes a truncated G protein exists. Interestingly, the majority sequence of strain J3666 was found to encode a G protein with an extended cytoplasmic tail, suggesting that there is the potential for considerable plasticity in the cytoplasmic tail of the G protein of PVM.

Recombinant respiratory syncytial virus with the G and F genes shifted to the promoter-proximal positions.

The genome of human respiratory syncytial virus (RSV) encodes 10 mRNAs and 11 proteins in the order 3'-NS1-NS2-N-P-M-SH-G-F-M2-1/M2-2-L-5'. The G and F glycoproteins are the major RSV neutralization and protective antigens. It seems likely that a high level of expression of G and F would be desirable for a live RSV vaccine. For mononegaviruses, the gene order is a major factor controlling the level of mRNA and protein expression due to the polar gradient of sequential transcription. In order to increase the expression of G and F, recombinant RSVs based on strain A2 were constructed in which the G or F gene was shifted from the sixth or seventh position (in a genome lacking the SH gene), respectively, to the first position (rRSV-G1/DeltaSH and rRSV-F1/DeltaSH, respectively). Another virus was made in which G and F were shifted together to the first and second positions, respectively (rRSV-G1F2/DeltaSH). Shifting one or two genes to the promoter-proximal position resulted in increased mRNA and protein expression of the shifted genes, with G and F expression increased up to 2.4-and 7.8-fold, respectively, at the mRNA level and approximately 2.5-fold at the protein level, compared to the parental virus. Interestingly, the transcription of downstream genes was not greatly affected even though shifting G or F, or G and F together, had the consequence of moving the block of genes NS1-NS2-N-P-M-(G) one or two positions further from the promoter. The efficiency of replication of the gene shift viruses in vitro was increased up to 10-fold. However, their efficiency of replication in the lower respiratory tracts of mice was statistically indistinguishable from that of the parental virus. In the upper respiratory tract, replication was slightly reduced on some days for viruses in which G was in the first position. The magnitude of the G-specific antibody response to the gene shift viruses was similar to that to the parental virus, whereas the F-specific response was increased up to fourfold, although this was not reflected in an increase of the neutralizing activity. Thus, shifting the G and F genes to the promoter-proximal position increased virus replication in vitro, had little effect on replication in the mouse, and increased the antigen-specific immunogenicity of the virus beyond that of parental RSV.