SARS-CoV-2 ORF6 disrupts innate immune signalling by inhibiting cellular mRNA export.

SARS-CoV-2 is a betacoronavirus and the etiological agent of COVID-19, a devastating infectious disease. Due to its far-reaching effect on human health, there is an urgent and growing need to understand the viral molecular biology of SARS-CoV-2 and its interaction with the host cell. SARS-CoV-2 encodes 9 predicted accessory proteins, which are presumed to be dispensable for in vitro replication, most likely having a role in modulating the host cell environment to aid viral replication. Here we show that the ORF6 accessory protein interacts with cellular Rae1 to inhibit cellular protein production by blocking mRNA export. We utilised cell fractionation coupled with mRNAseq to explore which cellular mRNA species are affected by ORF6 expression and show that ORF6 can inhibit the export of many mRNA including those encoding antiviral factors such as IRF1 and RIG-I. We also show that export of these mRNA is blocked in the context of SARS-CoV-2 infection. Together, our studies identify a novel mechanism by which SARS-CoV-2 can manipulate the host cell environment to supress antiviral responses, providing further understanding to the replication strategies of a virus that has caused an unprecedented global health crisis.

Virus restriction: Repurposing an essential cellular function to defend against viruses.

Eukaryotes are continually subjected to viral infections and, in response, have evolved a wide range of defence mechanisms. Two recent studies show how a duplicated copy of a cellular protein needed for cell growth and virus egress evolved to inhibit viruses while preserving cell viability.

Structure of a Ty1 restriction factor reveals the molecular basis of transposition copy number control.

Excessive replication of Saccharomyces cerevisiae Ty1 retrotransposons is regulated by Copy Number Control, a process requiring the p22/p18 protein produced from a sub-genomic transcript initiated within Ty1 GAG. In retrotransposition, Gag performs the capsid functions required for replication and re-integration. To minimize genomic damage, p22/p18 interrupts virus-like particle function by interaction with Gag. Here, we present structural, biophysical and genetic analyses of p18m, a minimal fragment of Gag that restricts transposition. The 2.8 Å crystal structure of p18m reveals an all α-helical protein related to mammalian and insect ARC proteins. p18m retains the capacity to dimerise in solution and the crystal structures reveal two exclusive dimer interfaces. We probe our findings through biophysical analysis of interface mutants as well as Ty1 transposition and p18m restriction in vivo. Our data provide insight into Ty1 Gag structure and suggest how p22/p18 might function in restriction through a blocking-of-assembly mechanism.

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells.

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis. This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Gv1, a Zinc Finger Gene Controlling Endogenous MLV Expression.

The genomes of inbred mice harbor around 50 endogenous murine leukemia virus (MLV) loci, although the specific complement varies greatly between strains. The Gv1 locus is known to control the transcription of endogenous MLVs and to be the dominant determinant of cell-surface presentation of MLV envelope, the GIX antigen. Here, we identify a single Krüppel-associated box zinc finger protein (ZFP) gene, Zfp998, as Gv1 and show it to be necessary and sufficient to determine the GIX+ phenotype. By long-read sequencing of bacterial artificial chromosome clones from 129 mice, the prototypic GIX+ strain, we reveal the source of sufficiency and deficiency as splice-acceptor variations and highlight the varying origins of the chromosomal region encompassing Gv1. Zfp998 becomes the second identified ZFP gene responsible for epigenetic suppression of endogenous MLVs in mice and further highlights the prominent role of this gene family in control of endogenous retroviruses.

Duplication and divergence of the retrovirus restriction gene Fv1 in Mus caroli allows protection from multiple retroviruses.

Viruses and their hosts are locked in an evolutionary race where resistance to infection is acquired by the hosts while viruses develop strategies to circumvent these host defenses. Forming one arm of the host defense armory are cell autonomous restriction factors like Fv1. Originally described as protecting laboratory mice from infection by murine leukemia virus (MLV), Fv1s from some wild mice have also been found to restrict non-MLV retroviruses, suggesting an important role in the protection against viruses in nature. We surveyed the Fv1 genes of wild mice trapped in Thailand and characterized their restriction activities against a panel of retroviruses. An extra copy of the Fv1 gene, named Fv7, was found on chromosome 6 of three closely related Asian species of mice: Mus caroli, M. cervicolor, and M. cookii. The presence of flanking repeats suggested it arose by LINE-mediated retroduplication within their most recent common ancestor. A high degree of natural variation was observed in both Fv1 and Fv7 and, on top of positive selection at certain residues, insertions and deletions were present that changed the length of the reading frames. These genes exhibited a range of restriction phenotypes, with activities directed against gamma-, spuma-, and lentiviruses. It seems likely, at least in the case of M. caroli, that the observed gene duplication may expand the breadth of restriction beyond the capacity of Fv1 alone and that one or more such viruses have recently driven or continue to drive the evolution of the Fv1 and Fv7 genes.

Antibody-induced internalisation of retroviral envelope glycoproteins is a signal initiation event.

As obligate parasites, viruses highjack, modify and repurpose the cellular machinery for their own replication. Viral proteins have, therefore, evolved biological functions, such as signalling potential, that alter host cell physiology in ways that are still incompletely understood. Retroviral envelope glycoproteins interact with several host proteins, extracellularly with their cellular receptor and anti-envelope antibodies, and intracellularly with proteins of the cytoskeleton or sorting, endocytosis and recirculation pathways. Here, we examined the impact of endogenous retroviral envelope glycoprotein expression and interaction with host proteins, particularly antibodies, on the cell, independently of retroviral infection. We found that in the commonly used C57BL/6 substrains of mice, where murine leukaemia virus (MLV) envelope glycoproteins are expressed by several endogenous MLV proviruses, the highest expressed MLV envelope glycoprotein is under the control of an immune-responsive cellular promoter, thus linking MLV envelope glycoprotein expression with immune activation. We further showed that antibody ligation induces extensive internalisation from the plasma membrane into endocytic compartments of MLV envelope glycoproteins, which are not normally subject to constitutive endocytosis. Importantly, antibody binding and internalisation of MLV envelope glycoproteins initiates signalling cascades in envelope-expressing murine lymphocytic cell lines, leading to cellular activation. Similar effects were observed by MLV envelope glycoprotein ligation by its cellular receptor mCAT-1, and by overexpression in human lymphocytic cells, where it required an intact tyrosine-based YXXΦ motif in the envelope glycoprotein cytoplasmic tail. Together, these results suggest that signalling potential is a general property of retroviral envelope glycoproteins and, therefore, a target for intervention.

Structure of Drosophila melanogaster ARC1 reveals a repurposed molecule with characteristics of retroviral Gag.

The tetrapod neuronal protein ARC and its Drosophila melanogaster homolog, dARC1, have important but differing roles in neuronal development. Both are thought to originate through exaptation of ancient Ty3/Gypsy retrotransposon Gag, with their novel function relying on an original capacity for self-assembly and encapsidation of nucleic acids. Here, we present the crystal structure of dARC1 CA and examine the relationship between dARC1, mammalian ARC, and the CA protein of circulating retroviruses. We show that while the overall architecture is highly related to that of orthoretroviral and spumaretroviral CA, there are substantial deviations in both amino- and carboxyl-terminal domains, potentially affecting recruitment of partner proteins and particle assembly. The degree of sequence and structural divergence suggests that Ty3/Gypsy Gag has been exapted on two separate occasions and that, although mammalian ARC and dARC1 share functional similarity, the structures have undergone different adaptations after appropriation into the tetrapod and insect genomes.

Structural basis for Fullerene geometry in a human endogenous retrovirus capsid.

The HML2 (HERV-K) group constitutes the most recently acquired family of human endogenous retroviruses, with many proviruses less than one million years old. Many maintain intact open reading frames and provirus expression together with HML2 particle formation are observed in early stage human embryo development and are associated with pluripotency as well as inflammatory disease, cancers and HIV-1 infection. Here, we reconstruct the core structural protein (CA) of an HML2 retrovirus, assemble particles in vitro and employ single particle cryogenic electron microscopy (cryo-EM) to determine structures of four classes of CA Fullerene shell assemblies. These icosahedral and capsular assemblies reveal at high-resolution the molecular interactions that allow CA to form both pentamers and hexamers and show how invariant pentamers and structurally plastic hexamers associate to form the unique polyhedral structures found in retroviral cores.

LTR retroelement expansion of the human cancer transcriptome and immunopeptidome revealed by de novo transcript assembly.

Dysregulated endogenous retroelements (EREs) are increasingly implicated in the initiation, progression, and immune surveillance of human cancer. However, incomplete knowledge of ERE activity limits mechanistic studies. By using pan-cancer de novo transcript assembly, we uncover the extent and complexity of ERE transcription. The current assembly doubled the number of previously annotated transcripts overlapping with long-terminal repeat (LTR) elements, several thousand of which were expressed specifically in one or a few related cancer types. Exemplified in melanoma, LTR-overlapping transcripts were highly predictable, disease prognostic, and closely linked with molecularly defined subtypes. They further showed the potential to affect disease-relevant genes, as well as produce novel cancer-specific antigenic peptides. This extended view of LTR elements provides the framework for functional validation of affected genes and targets for cancer immunotherapy.

Positive selection in dNTPase SAMHD1 throughout mammalian evolution.

The vertebrate protein SAMHD1 is highly unusual in having roles in cellular metabolic regulation, antiviral restriction, and regulation of innate immunity. Its deoxynucleoside triphosphohydrolase activity regulates cellular dNTP concentration, reducing levels below those required by lentiviruses and other viruses to replicate. To counter this threat, some primate lentiviruses encode accessory proteins that bind SAMHD1 and induce its degradation; in turn, positive diversifying selection has been observed in regions bound by these lentiviral proteins, suggesting that primate SAMHD1 has coevolved to evade these countermeasures. Moreover, deleterious polymorphisms in human SAMHD1 are associated with autoimmune disease linked to uncontrolled DNA synthesis of endogenous retroelements. Little is known about how evolutionary pressures affect these different SAMHD1 functions. Here, we examine the deeper history of these interactions by testing whether evolutionary signatures in SAMHD1 extend to other mammalian groups and exploring the molecular basis of this coevolution. Using codon-based likelihood models, we find positive selection in SAMHD1 within each mammal lineage for which sequence data are available. We observe positive selection at sites clustered around T592, a residue that is phosphorylated to regulate SAMHD1 activity. We verify experimentally that mutations within this cluster affect catalytic rate and lentiviral restriction, suggesting that virus-host coevolution has required adaptations of enzymatic function. Thus, persistent positive selection may have involved the adaptation of SAMHD1 regulation to balance antiviral, metabolic, and innate immunity functions.

A helical LC3-interacting region mediates the interaction between the retroviral restriction factor Trim5α and mammalian autophagy-related ATG8 proteins.

The retroviral restriction factor tripartite motif-containing 5α (Trim5α) acts during the early postentry stages of the retroviral life cycle to block infection by a broad range of retroviruses, disrupting reverse transcription and integration. The mechanism of this restriction is poorly understood, but it has recently been suggested to involve recruitment of components of the autophagy machinery, including members of the mammalian autophagy-related 8 (ATG8) family involved in targeting proteins to the autophagosome. To better understand the molecular details of this interaction, here we utilized analytical ultracentrifugation to characterize the binding of six ATG8 isoforms and determined the crystal structure of the Trim5α Bbox coiled-coil region in complex with one member of the mammalian ATG8 proteins, autophagy-related protein LC3 B (LC3B). We found that Trim5α binds all mammalian ATG8s and that, unlike the typical LC3-interacting region (LIR) that binds to mammalian ATG8s through a ß-strand motif comprising approximately six residues, LC3B binds to Trim5α via the α-helical coiled-coil region. The orientation of the structure demonstrated that LC3B could be accommodated within a Trim5α assembly that can bind the retroviral capsid. However, mutation of the binding interface does not affect retroviral restriction. Comparison of the typical linear ß-strand LIR with our atypical helical LIR reveals a conservation of the presentation of residues that are required for the interaction with LC3B. This observation expands the range of LC3B-binding proteins to include helical binding motifs and demonstrates a link between Trim5α and components of the autophagosome.

Evolutionary journey of the retroviral restriction gene Fv1.

Both exogenous and endogenous retroviruses have long been studied in mice, and some of the earliest mouse studies focused on the heritability of genetic factors influencing permissivity and resistance to infection. The prototypic retroviral restriction factor, Fv1, is now understood to exhibit a degree of control across multiple retroviral genera and is highly diverse within Mus To better understand the age and evolutionary history of Fv1, a comprehensive survey of the Muroidea was conducted, allowing the progenitor integration to be dated to ∼45 million years. Intact coding potential is visible beyond Mus, and sequence analysis reveals strong signatures of positive selection also within field mice, ApodemusFv1's survival for such a period implies a recurring and shifting retroviral burden imparting the necessary selective pressures-an influence likely also common to analogous factors. Regions of Fv1 adapt cooperatively, highlighting its preference for repeated structures and suggesting that this functionally constrained aspect of the retroviral capsid lattice presents a common target in the evolution of intrinsic immunity.

Restoration of Endogenous Retrovirus Infectivity Impacts Mouse Cancer Models.

Mouse models have been instrumental in establishing fundamental principles of cancer initiation and progression and continue to be invaluable in the discovery and further development of cancer therapies. Nevertheless, important aspects of human disease are imperfectly approximated in mouse models, notably the involvement of endogenous retroviruses (ERVs). Replication-defective ERVs, present in both humans and mice, may affect tumor development and antitumor immunity through mechanisms not involving infection. Here, we revealed an adverse effect of murine ERVs with restored infectivity on the behavior of mouse cancer models. In contrast to human cancer, where infectious ERVs have never been detected, we found that ERV infectivity was frequently restored in transplantable, as well as genetic, mouse cancer models. Such replication-competent, ERV-derived retroviruses were responsible for unusually high expression of retroviral nucleic acids and proteins in mouse cancers. Infectious ERV-derived retroviruses produced by mouse cancer cells could directly infect tumor-infiltrating host immune cells and fundamentally modified the host's immune defenses to cancer, as well as the outcome of immunotherapy. Therefore, infectious retroviruses, variably arising in mouse cancer models, but not in human cancer, have the potential to confound many immunologic studies and should be considered as a variable, if not altogether avoided. Cancer Immunol Res; 6(11); 1292-300. ©2018 AACR.

Making a virtue of necessity: the pleiotropic role of human endogenous retroviruses in cancer.

Like all other mammals, humans harbour an astonishing number of endogenous retroviruses (ERVs), as well as other retroelements, embedded in their genome. These remnants of ancestral germline infection with distinct exogenous retroviruses display various degrees of open reading frame integrity and replication capability. Modern day exogenous retroviruses, as well as the infectious predecessors of ERVs, are demonstrably oncogenic. Further, replication-competent ERVs continue to cause cancers in many other species of mammal. Moreover, human cancers are characterized by transcriptional activation of human endogenous retroviruses (HERVs). These observations conspire to incriminate HERVs as causative agents of human cancer. However, exhaustive investigation of cancer genomes suggests that HERVs have entirely lost the ability for re-infection and thus the potential for insertional mutagenic activity. Although there may be non-insertional mechanisms by which HERVs contribute to cancer development, recent evidence also uncovers potent anti-tumour activities exerted by HERV replication intermediates or protein products. On balance, it appears that HERVs, despite their oncogenic past, now represent potential targets for immune-mediated anti-tumour mechanisms.This article is part of the themed issue 'Human oncogenic viruses'.

Mutation of the Putative Immunosuppressive Domain of the Retroviral Envelope Glycoprotein Compromises Infectivity.

The envelope glycoprotein of diverse endogenous and exogenous retroviruses is considered inherently immunosuppressive. Extensive work mapped the immunosuppressive activity to a highly conserved domain, termed the immunosuppressive domain (ISD), in the transmembrane (TM) subunit of the envelope glycoprotein and identified two naturally polymorphic key residues that afford immunosuppressive activity to distinct envelope glycoproteins. Concurrent mutation of these two key residues (E14R and A20F) in the envelope glycoprotein of the Friend murine leukemia virus (F-MLV) ISD has been reported to abolish its immunosuppressive activity, without affecting its fusogenicity, and to weaken the ability of the virus to replicate specifically in immunocompetent hosts. Here, we show that mutation of these key residues did, in fact, result in a substantial loss of F-MLV infectivity, independently of host immunity, challenging whether associations exist between the two. Notably, a loss of infectivity incurred by the F-MLV mutant with the E14R and A20F double ISD mutation was conditional on expression of the ecotropic envelope receptor murine cationic amino acid transporter-1 (mCAT1) in the virus-producing cell. Indeed, the F-MLV mutant retained infectivity when it was produced by human cells, which naturally lack mCAT1 expression, but not by murine cells. Furthermore, mCAT1 overexpression in human cells impaired the infectivity of both the F-MLV double mutant and the wild-type F-MLV strain, suggesting a finely tuned relationship between the levels of mCAT1 in the producer cell and the infectivity of the virions produced. An adverse effect on this relationship, rather than disruption of the putative ISD, is therefore more likely to explain the loss of F-MLV infectivity incurred by mutations in key ISD residues E14 and A20.IMPORTANCE Retroviruses can interact with their hosts in ways that, although not entirely understood, can greatly influence their pathogenic potential. One such example is a putative immunosuppressive activity, which has been mapped to a conserved domain of the retroviral envelope glycoprotein of several exogenous as well as endogenous retroviruses. In this study, mutations naturally found in some envelope glycoproteins lacking immunosuppressive activity were shown to affect retrovirus infectivity only if the host cell that produced the retrovirus also expressed the cellular entry receptor. These findings shed light on a novel role for this conserved domain in providing the necessary stability to the envelope glycoprotein in order to withstand the interaction with the cellular receptor during virus formation. This function of the domain is critical for further elucidation of the mechanism of immunosuppression mediated by the retroviral envelope glycoprotein.

A comparative analysis of the foamy and ortho virus capsid structures reveals an ancient domain duplication.

BACKGROUND: The Spumaretrovirinae (foamy viruses) and the Orthoretrovirinae (e.g. HIV) share many similarities both in genome structure and the sequences of the core viral encoded proteins, such as the aspartyl protease and reverse transcriptase. Similarity in the gag region of the genome is less obvious at the sequence level but has been illuminated by the recent solution of the foamy virus capsid (CA) structure. This revealed a clear structural similarity to the orthoretrovirus capsids but with marked differences that left uncertainty in the relationship between the two domains that comprise the structure. METHODS: We have applied protein structure comparison methods in order to try and resolve this ambiguous relationship. These included both the DALI method and the SAP method, with rigorous statistical tests applied to the results of both methods. For this, we employed collections of artificial fold 'decoys' (generated from the pair of native structures being compared) to provide a customised background distribution for each comparison, thus allowing significance levels to be estimated. RESULTS: We have shown that the relationship of the two domains conforms to a simple linear correspondence rather than a domain transposition. These similarities suggest that the origin of both viral capsids was a common ancestor with a double domain structure. In addition, we show that there is also a significant structural similarity between the amino and carboxy domains in both the foamy and ortho viruses. CONCLUSIONS: These results indicate that, as well as the duplication of the double domain capsid, there may have been an even more ancient gene-duplication that preceded the double domain structure. In addition, our structure comparison methodology demonstrates a general approach to problems where the components have a high intrinsic level of similarity.

SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells.

SAMHD1 is an intracellular enzyme that specifically degrades deoxynucleoside triphosphates into component nucleoside and inorganic triphosphate. In myeloid-derived dendritic cells and macrophages as well as resting T-cells, SAMHD1 blocks HIV-1 infection through this dNTP triphosphohydrolase activity by reducing the cellular dNTP pool to a level that cannot support productive reverse transcription. We now show that, in addition to this direct effect on virus replication, manipulating cellular SAMHD1 activity can significantly enhance or decrease the anti-HIV-1 efficacy of nucleotide analogue reverse transcription inhibitors presumably as a result of modulating dNTP pools that compete for recruitment by viral polymerases. Further, a variety of other nucleotide-based analogues, not normally considered antiretrovirals, such as the anti-herpes drugs Aciclovir and Ganciclovir and the anti-cancer drug Clofarabine are now revealed as potent anti-HIV-1 agents, under conditions of low dNTPs. This in turn suggests novel uses for nucleotide analogues to inhibit HIV-1 in differentiated cells low in dNTPs.

Physiological and Pathological Transcriptional Activation of Endogenous Retroelements Assessed by RNA-Sequencing of B Lymphocytes.

In addition to evolutionarily-accrued sequence mutation or deletion, endogenous retroelements (EREs) in eukaryotic genomes are subject to epigenetic silencing, preventing or reducing their transcription, particularly in the germplasm. Nevertheless, transcriptional activation of EREs, including endogenous retroviruses (ERVs) and long interspersed nuclear elements (LINEs), is observed in somatic cells, variably upon cellular differentiation and frequently upon cellular transformation. ERE transcription is modulated during physiological and pathological immune cell activation, as well as in immune cell cancers. However, our understanding of the potential consequences of such modulation remains incomplete, partly due to the relative scarcity of information regarding genome-wide ERE transcriptional patterns in immune cells. Here, we describe a methodology that allows probing RNA-sequencing (RNA-seq) data for genome-wide expression of EREs in murine and human cells. Our analysis of B cells reveals that their transcriptional response during immune activation is dominated by induction of gene transcription, and that EREs respond to a much lesser extent. The transcriptional activity of the majority of EREs is either unaffected or reduced by B cell activation both in mice and humans, albeit LINEs appear considerably more responsive in the latter host. Nevertheless, a small number of highly distinct ERVs are strongly and consistently induced during B cell activation. Importantly, this pattern contrasts starkly with B cell transformation, which exhibits widespread induction of EREs, including ERVs that minimally overlap with those responsive to immune stimulation. The distinctive patterns of ERE induction suggest different underlying mechanisms and will help separate physiological from pathological expression.