The envelope gene of transmitted HIV-1 resists a late interferon gamma-induced block

Type I interferon (IFN) signaling engenders an antiviral state that likely plays an important role in constraining HIV-1 transmission and contributes to defining subsequent AIDS pathogenesis. Type II IFN (IFNγ) also induces an antiviral state but is often primarily considered to be an immunomodulatory cytokine. We report that IFNγ stimulation can induce an antiviral state that can be both distinct from that of type I interferon, and can potently inhibit HIV-1 in primary CD4+ T cells and a number of human cell lines. Strikingly, we find that transmitted/founder (TF) HIV-1 viruses can resist a late block that is induced by type II IFN, and the use of chimeric IFNγ- sensitive/resistant viruses indicates that interferon-resistance maps to the env gene. Simultaneously, in vitro evolution also revealed that just a single amino acid substitution in envelope can confer substantial resistance to IFN-mediated inhibition. Thus, the env gene of transmitted HIV-1 confers resistance to a late block that is phenotypically distinct from those previously described to be resisted by env, and is therefore mediated by unknown IFNγ-stimulated factor(s) in human CD4+ T cells and cell lines. This important unidentified block could play a key role in constraining HIV-1 transmission

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods

The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity

Disrupted Peyer’s Patch Microanatomy in COVID-19 Including Germinal Centre Atrophy Independent of Local Virus

Confirmed SARS-coronavirus-2 infection with gastrointestinal symptoms and changes in microbiota associated with coronavirus disease 2019 (COVID-19) severity have been previously reported, but the disease impact on the architecture and cellularity of ileal Peyer’s patches (PP) remains unknown. Here we analysed post-mortem tissues from throughout the gastrointestinal (GI) tract of patients who died with COVID-19. When virus was detected by PCR in the GI tract, immunohistochemistry identified virus in epithelium and lamina propria macrophages, but not in lymphoid tissues. Immunohistochemistry and imaging mass cytometry (IMC) analysis of ileal PP revealed depletion of germinal centres (GC), disruption of B cell/T cell zonation and decreased potential B and T cell interaction and lower nuclear density in COVID-19 patients. This occurred independent of the local viral levels. The changes in PP demonstrate that the ability to mount an intestinal immune response is compromised in severe COVID-19, which could contribute to observed dysbiosis

Sensitivity to BST-2 restriction correlates with Orthobunyavirus host range

Orthobunyaviruses include several recently emerging viruses of significant medical and veterinary importance. There is currently very limited understanding on what determines the host species range of these pathogens. In this study we discovered that BST-2/tetherin restricts orthobunyavirus replication in a host-specific manner. We show that viruses with human tropism (Oropouche virus and La Crosse virus) are restricted by sheep BST-2 but not by the human orthologue, while viruses with ruminant tropism (Schmallenberg virus and others) are restricted by human BST-2 but not by the sheep orthologue. We also show that BST-2 blocks orthobunyaviruses replication by reducing the amount of envelope glycoprotein into viral particles egressing from infected cells. This is the first study identifying a restriction factor that correlates with species susceptibility to orthobunyavirus infection. This work provides insight to help us dissect the adaptive changes that bunyaviruses require to cross the species barrier and emerge into new species

The origins of SARS-CoV-2: a critical review

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a “laboratory escape” scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2

Targeted Restriction of Viral Gene Expression and Replication by the ZAP Antiviral System

The zinc finger antiviral protein (ZAP) restricts the replication of a broad range of RNA and DNA viruses. ZAP directly binds viral RNA, targeting it for degradation and inhibiting its translation. While the full scope of RNA determinants involved in mediating selective ZAP activity is unclear, ZAP binds CpG dinucleotides, dictating at least part of its target specificity. ZAP interacts with many cellular proteins, although only a few have been demonstrated to be essential for its antiviral activity, including the 3 prime -5 prime exoribonuclease exosome complex, TRIM25, and KHNYN. In addition to inhibiting viral gene expression, ZAP also directly and indirectly targets a subset of cellular messenger RNAs to regulate the innate immune response. Overall, ZAP protects a cell from viral infection by restricting viral replication and regulating cellular gene expression. Further understanding of the ZAP antiviral system may allow for novel viral vaccine and anticancer therapy development.</p

Homebrew:Protocol for glassmilk-based nucleic-acid extraction for SARS-CoV-2 diagnostics

The gold standard protocol for SARS-CoV-2 infection detection remains reverse transcription quantitative polymerase chain reaction (RT-qPCR), which detects viral RNA more sensitively than any other approach. Here, we present Homebrew, a low cost protocol to extract RNA using widely available reagents. Homebrew is as sensitive as commercially available RNA extraction kits. Homebrew allows for sample pooling and can be adapted for automation in high throughput settings

Sars-cov-2 is restricted by zinc finger antiviral protein despite preadaptation to the low-cpg environment in humans

ABSTRACT Recent evidence shows that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is sensitive to interferons (IFNs). However, the most effective types of IFNs and the underlying antiviral effectors remain to be defined. Here, we show that zinc finger antiviral protein (ZAP), which preferentially targets CpG dinucleotides in viral RNA sequences, restricts SARS-CoV-2. We further 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. Comprehensive sequence analyses revealed that SARS-CoV-2 and its closest relatives from horseshoe bats showed the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, endogenous ZAP expression restricted SARS-CoV-2 replication in human lung cells, particularly upon treatment with IFN-α or IFN-γ. Both the long and the short isoforms of human ZAP reduced SARS-CoV-2 RNA expression levels, but the former did so with greater efficiency. Finally, we show that the ability to restrict SARS-CoV-2 is conserved in ZAP orthologues of the reservoir bat and potential intermediate pangolin hosts of human coronaviruses. Altogether, our results show that ZAP is an important effector of the innate response against SARS-CoV-2, although this pandemic pathogen emerged from zoonosis of a coronavirus that was preadapted to the low-CpG environment in humans. IMPORTANCE Although interferons inhibit SARS-CoV-2 and have been evaluated for treatment of coronavirus disease 2019 (COVID-19), the most effective types and antiviral effectors remain to be defined. Here, we show that IFN-γ is particularly potent in restricting SARS-CoV-2 and in inducing expression of the antiviral factor ZAP in human lung cells. Knockdown experiments revealed that endogenous ZAP significantly restricts SARS-CoV-2. We further show that CpG dinucleotides which are specifically targeted by ZAP are strongly suppressed in the SARS-CoV-2 genome and that the two closest horseshoe bat relatives of SARS-CoV-2 show the lowest genomic CpG content of all coronavirus sequences available from this reservoir host. Nonetheless, both the short and long isoforms of human ZAP reduced SARS-CoV-2 RNA levels, and this activity was conserved in horseshoe bat and pangolin ZAP orthologues. Our findings indicating that type II interferon is particularly efficient against SARS-CoV-2 and that ZAP restricts this pandemic viral pathogen might promote the development of effective immune therapies against COVID-19