Available upon all requests? How and why we should better incentivize the sharing of biomaterials.

Biomaterial sharing offers enormous benefits for research and for the scientific community. Individuals, funders, institutions, and journals can overcome the barriers to sharing and work together to promote a better sharing culture.

The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness.

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced 'antiviral state', we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be.

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction.

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

The antiviral state has shaped the CpG composition of the vertebrate interferome to avoid self-targeting.

Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.

A prenylated dsRNA sensor protects against severe COVID-19.

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

TRIM69 Inhibits Vesicular Stomatitis Indiana Virus.

Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.

Interferon-Stimulated Gene (ISG)-Expression Screening Reveals the Specific Antibunyaviral Activity of ISG20.

Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an "antiviral state." Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ∼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

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 (IFN-γ) also induces an antiviral state but is often primarily considered to be an immunomodulatory cytokine. We report that IFN-γ 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 IFN-γ-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 the 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 blocks previously described to be resisted by env and is therefore mediated by unknown IFN-γ-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.IMPORTANCE The human immune system can hinder invading pathogens through interferon (IFN) signaling. One consequence of this signaling is that cells enter an antiviral state, increasing the levels of hundreds of defenses that can inhibit the replication and spread of viruses. The majority of HIV-1 infections result from a single virus particle (the transmitted/founder) that makes it past these defenses and colonizes the host. Thus, the founder virus is hypothesized to be a relatively interferon-resistant entity. Here, we show that certain HIV-1 envelope genes have the unanticipated ability to resist specific human defenses mediated by different types of interferons. Strikingly, the envelope gene from a founder HIV-1 virus is far better at evading these defenses than the corresponding gene from a common HIV-1 lab strain. Thus, these defenses could play a role in constraining the transmission of HIV-1 and may select for transmitted viruses that are resistant to this IFN-mediated inhibition.

Identification of Interferon-Stimulated Genes with Antiretroviral Activity.

Interferons (IFNs) exert their anti-viral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). The activity of known ISGs is insufficient to account for the antiretroviral effects of IFN, suggesting that ISGs with antiretroviral activity are yet to be described. We constructed an arrayed library of ISGs from rhesus macaques and tested the ability of hundreds of individual macaque and human ISGs to inhibit early and late replication steps for 11 members of the retroviridae from various host species. These screens uncovered numerous ISGs with antiretroviral activity at both the early and late stages of virus replication. Detailed analyses of two antiretroviral ISGs indicate that indoleamine 2,3-dioxygenase 1 (IDO1) can inhibit retroviral replication by metabolite depletion while tripartite motif-56 (TRIM56) accentuates ISG induction by IFNα and inhibits the expression of late HIV-1 genes. Overall, these studies reveal numerous host proteins that mediate the antiretroviral activity of IFNs.

Uneven genetic robustness of HIV-1 integrase.

UNLABELLED: Genetic robustness (tolerance of mutation) may be a naturally selected property in some viruses, because it should enhance adaptability. Robustness should be especially beneficial to viruses like HIV-1 that exhibit high mutation rates and exist in immunologically hostile environments. Surprisingly, however, the HIV-1 capsid protein (CA) exhibits extreme fragility. To determine whether fragility is a general property of HIV-1 proteins, we created a large library of random, single-amino-acid mutants in HIV-1 integrase (IN), covering >40% of amino acid positions. Despite similar degrees of sequence variation in naturally occurring IN and CA sequences, we found that HIV-1 IN was significantly more robust than CA, with random nonsilent IN mutations only half as likely to cause lethal defects. Interestingly, IN and CA were similar in that a subset of mutations with high in vitro fitness were rare in natural populations. IN mutations of this type were more likely to occur in the buried interior of the modeled HIV-1 intasome, suggesting that even very subtle fitness effects suppress variation in natural HIV-1 populations. Lethal mutations, in particular those that perturbed particle production, proteolytic processing, and particle-associated IN levels, were strikingly localized at specific IN subunit interfaces. This observation strongly suggests that binding interactions between particular IN subunits regulate proteolysis during HIV-1 virion morphogenesis. Overall, use of the IN mutant library in conjunction with structural models demonstrates the overall robustness of IN and highlights particular regions of vulnerability that may be targeted in therapeutic interventions. IMPORTANCE: The HIV-1 integrase (IN) protein is responsible for the integration of the viral genome into the host cell chromosome. To measure the capacity of IN to maintain function in the face of mutation, and to probe structure/function relationships, we created a library of random single-amino-acid IN mutations that could mimic the types of mutations that naturally occur during HIV-1 infection. Previously, we measured the robustness of HIV-1 capsid in this manner and determined that it is extremely intolerant of mutation. In contrast to CA, HIV-1 IN proved relatively robust, with far fewer mutations causing lethal defects. However, when we subsequently mapped the lethal mutations onto a model of the structure of the multisubunit IN-viral DNA complex, we found the lethal mutations that caused virus morphogenesis defects tended to be highly localized at subunit interfaces. This discovery of vulnerable regions of HIV-1 IN could inform development of novel therapeutics.

Host and viral determinants of Mx2 antiretroviral activity.

Myxovirus resistance 2 (Mx2/MxB) has recently been uncovered as an effector of the anti-HIV-1 activity of type I interferons (IFNs) that inhibits HIV-1 at an early stage postinfection, after reverse transcription but prior to proviral integration into host DNA. The mechanistic details of Mx2 antiviral activity are not yet understood, but a few substitutions in the HIV-1 capsid have been shown to confer resistance to Mx2. Through a combination of in vitro evolution and unbiased mutagenesis, we further map the determinants of sensitivity to Mx2 and reveal that multiple capsid (CA) surfaces define sensitivity to Mx2. Intriguingly, we reveal an unanticipated sensitivity determinant within the C-terminal domain of capsid. We also report that Mx2s derived from multiple primate species share the capacity to potently inhibit HIV-1, whereas selected nonprimate orthologs have no such activity. Like TRIM5α, another CA targeting antiretroviral protein, primate Mx2s exhibit species-dependent variation in antiviral specificity against at least one extant virus and multiple HIV-1 capsid mutants. Using a combination of chimeric Mx2 proteins and evolution-guided approaches, we reveal that a single residue close to the N terminus that has evolved under positive selection can determine antiviral specificity. Thus, the variable N-terminal region can define the spectrum of viruses inhibited by Mx2. Importance: Type I interferons (IFNs) inhibit the replication of most mammalian viruses. IFN stimulation upregulates hundreds of different IFN-stimulated genes (ISGs), but it is often unclear which ISGs are responsible for inhibition of a given virus. Recently, Mx2 was identified as an ISG that contributes to the inhibition of HIV-1 replication by type I IFN. Thus, Mx2 might inhibit HIV-1 replication in patients, and this inhibitory action might have therapeutic potential. The mechanistic details of how Mx2 inhibits HIV-1 are currently unclear, but the HIV-1 capsid protein is the likely viral target. Here, we determine the regions of capsid that specify sensitivity to Mx2. We demonstrate that Mx2 from multiple primates can inhibit HIV-1, whereas Mx2 from other mammals (dogs and sheep) cannot. We also show that primate variants of Mx2 differ in the spectrum of lentiviruses they inhibit and that a single residue in Mx2 can determine this antiviral specificity.

Extreme genetic fragility of the HIV-1 capsid.

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies.