A conserved opal termination codon optimizes a temperature-dependent tradeoff between protein production and processing in alphaviruses.

Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that often require transmission between arthropod and vertebrate hosts for their sustained propagation. Most alphaviruses encode an opal (UGA) termination codon in nonstructural protein 3 (nsP3) upstream of the viral polymerase, nsP4. The selective constraints underlying the conservation of the opal codon are poorly understood. Using primate and mosquito cells, we explored the role and selective pressure on the nsP3 opal codon through extensive mutational analysis in the prototype alphavirus, Sindbis virus (SINV). We found that the opal codon is highly favored over all other codons in primate cells under native 37ºC growth conditions. However, this preference is diminished in mosquito and primate cells grown at a lower temperature. Thus, the primary determinant driving the selection of the opal stop codon is not host genetics but the passaging temperature. We show that the opal codon is preferred over amber and ochre termination codons because it results in the highest translational readthrough and polymerase production. However, substituting the opal codon with sense codons leads to excessive full-length polyprotein (P1234) production, which disrupts optimal nsP polyprotein processing, delays the switch from minus-strand to positive-strand RNA production, and significantly reduces SINV fitness at 37°C; this fitness defect is relieved at lower temperatures. A naturally occurring suppressor mutation unexpectedly compensates for a delayed transition from minus to genomic RNA production by also delaying the subsequent transition between genomic and sub-genomic RNA production. Our study reveals that the opal stop codon is the best solution for alphavirus replication at 37ºC, producing enough nsP4 protein to maximize replication without disrupting nsP processing and RNA replication transitions needed for optimal fitness. Our study uncovers the intricate strategy dual-host alphaviruses use at a single codon to optimize fitness.

CARD8 inflammasome activation during HIV-1 cell-to-cell transmission.

Our previous work demonstrated that CARD8 detects HIV-1 infection by sensing the enzymatic activity of the HIV protease, resulting in CARD8-dependent inflammasome activation (Kulsuptrakul et al., 2023). CARD8 recognition of HIV-1 protease activity is conferred by a HIV protease substrate mimic within the CARD8 N-terminus, which when cleaved by HIV protease triggers CARD8 inflammasome activation. Here, we sought to understand CARD8 responses to HIV-1 when the virus is transmitted through cell-to-cell infection from infected cells to target cells via a viral synapse. We observed that cell-to-cell transmission of HIV-1 induces CARD8 inflammasome activation in immortalized cells and primary human monocyte-derived macrophages in a manner that is dependent on viral protease activity and largely independent of the NLRP3 inflammasome. Additionally, to further evaluate the viral determinants of CARD8 sensing, we tested a panel of HIV protease inhibitor resistant clones to establish how variation in HIV protease affects CARD8 activation. We identified mutant HIV-1 proteases that differentially cleave and activate CARD8 compared to wildtype HIV-1, thus indicating that natural variation in HIV protease affects not only the cleavage of the viral Gag-Pol polyprotein but also likely impacts innate sensing and inflammation.

Antiviral Mx proteins have an ancient origin and widespread distribution among eukaryotes.

First identified in mammals, Mx proteins are potent antivirals against a broad swathe of viruses. Mx proteins arose within the Dynamin superfamily of proteins (DSP), mediating critical cellular processes, such as endocytosis and mitochondrial, plastid, and peroxisomal dynamics. And yet, the evolutionary origins of Mx proteins are poorly understood. Using a series of phylogenomic analyses with stepwise increments in taxonomic coverage, we show that Mx proteins predate the interferon signaling system in vertebrates. Our analyses find an ancient monophyletic DSP lineage in eukaryotes that groups vertebrate and invertebrate Mx proteins with previously undescribed fungal MxF proteins, the relatively uncharacterized plant and algal Dynamin 4A/4C proteins, and representatives from several early-branching eukaryotic lineages. Thus, Mx-like proteins date back close to the origin of Eukarya. Our phylogenetic analyses also reveal that host-encoded and NCLDV (nucleocytoplasmic large DNA viruses)-encoded DSPs are interspersed in four distinct DSP lineages, indicating recurrent viral theft of host DSPs. Our analyses thus reveal an ancient history of viral and antiviral functions encoded by the Dynamin superfamily in eukaryotes.

A targeted CRISPR screen identifies ETS1 as a regulator of HIV latency.

Human Immunodeficiency virus (HIV) infection is regulated by a wide array of host cell factors that combine to influence viral transcription and latency. To understand the complex relationship between the host cell and HIV latency, we performed a lentiviral CRISPR screen that targeted a set of host cell genes whose expression or activity correlates with HIV expression. We further investigated one of the identified factors - the transcription factor ETS1 and found that it is required for maintenance of HIV latency in a primary CD4 T cell model. Interestingly, ETS1 played divergent roles in actively infected and latently infected CD4 T cells, with knockout of ETS1 leading to reduced HIV expression in actively infected cells, but increased HIV expression in latently infected cells, indicating that ETS1 can play both a positive and negative role in HIV expression. CRISPR/Cas9 knockout of ETS1 in CD4 T cells from ART-suppressed people with HIV (PWH) confirmed that ETS1 maintains transcriptional repression of the clinical HIV reservoir. Transcriptomic profiling of ETS1-depleted cells from PWH identified a set of host cell pathways involved in viral transcription that are controlled by ETS1 in resting CD4 T cells. In particular, we observed that ETS1 knockout increased expression of the long non-coding RNA MALAT1 that has been previously identified as a positive regulator of HIV expression. Furthermore, the impact of ETS1 depletion on HIV expression in latently infected cells was partially dependent on MALAT1. Overall, these data demonstrate that ETS1 is an important regulator of HIV latency and influences expression of several cellular genes, including MALAT1, that could have a direct or indirect impact on HIV expression. Author Summary: HIV latency is a major obstacle for the eradication of HIV. However, molecular mechanisms that restrict proviral expression during therapy are not well understood. Identification of host cell factors that silence HIV would create opportunities for targeting these factors to reverse latency and eliminate infected cells. Our study aimed to explore mechanisms of latency in infected cells by employing a lentiviral CRISPR screen and CRISPR/Cas9 knockout in primary CD4 T cells. These experiments revealed that ETS1 is essential for maintaining HIV latency in primary CD4 T cells and we further confirmed ETS1's role in maintaining HIV latency through CRISPR/Cas9 knockout in CD4 T cells from antiretroviral therapy (ART)-suppressed individuals with HIV. Transcriptomic profiling of ETS1-depleted cells from these individuals identified several host cell pathways involved in viral transcription regulated by ETS1, including the long non-coding RNA MALAT1. Overall, our study demonstrates that ETS1 is a critical regulator of HIV latency, affecting the expression of several cellular genes that directly or indirectly influence HIV expression.

Indels allow antiviral proteins to evolve functional novelty inaccessible by missense mutations.

Antiviral proteins often evolve rapidly at virus-binding interfaces to defend against new viruses. We investigated whether antiviral adaptation via missense mutations might face limits, which insertion or deletion mutations (indels) could overcome. We report one such case of a nearly insurmountable evolutionary challenge: the human anti-retroviral protein TRIM5α requires more than five missense mutations in its specificity-determining v1 loop to restrict a divergent simian immunodeficiency virus (SIV). However, duplicating just one amino acid in v1 enables human TRIM5α to potently restrict SIV in a single evolutionary step. Moreover, natural primate TRIM5α v1 loops have evolved indels that confer novel antiviral specificities. Thus, indels enable antiviral proteins to overcome viral challenges inaccessible by missense mutations, revealing the potential of these often-overlooked mutations in driving protein innovation.

Capsid-dependent lentiviral restrictions.

Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as Lv1 through Lv5, have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

A CRISPR Screen of HIV Dependency Factors Reveals That CCNT1 Is Non-Essential in T Cells but Required for HIV-1 Reactivation from Latency.

We sought to explore the hypothesis that host factors required for HIV-1 replication also play a role in latency reversal. Using a CRISPR gene library of putative HIV dependency factors, we performed a screen to identify genes required for latency reactivation. We identified several HIV-1 dependency factors that play a key role in HIV-1 latency reactivation including ELL, UBE2M, TBL1XR1, HDAC3, AMBRA1, and ALYREF. The knockout of Cyclin T1 (CCNT1), a component of the P-TEFb complex that is important for transcription elongation, was the top hit in the screen and had the largest effect on HIV latency reversal with a wide variety of latency reversal agents. Moreover, CCNT1 knockout prevents latency reactivation in a primary CD4+ T cell model of HIV latency without affecting the activation of these cells. RNA sequencing data showed that CCNT1 regulates HIV-1 proviral genes to a larger extent than any other host gene and had no significant effects on RNA transcripts in primary T cells after activation. We conclude that CCNT1 function is non-essential in T cells but is absolutely required for HIV latency reversal.

A CRISPR screen of HIV dependency factors reveals CCNT1 is non-essential in T cells but required for HIV-1 reactivation from latency.

We sought to explore the hypothesis that host factors required for HIV-1 replication also play a role in latency reversal. Using a CRISPR gene library of putative HIV dependency factors, we performed a screen to identify genes required for latency reactivation. We identified several HIV-1 dependency factors that play a key role in HIV-1 latency reactivation including ELL , UBE2M , TBL1XR1 , HDAC3 , AMBRA1 , and ALYREF . Knockout of Cyclin T1 ( CCNT1 ), a component of the P-TEFb complex important for transcription elongation, was the top hit in the screen and had the largest effect on HIV latency reversal with a wide variety of latency reversal agents. Moreover, CCNT1 knockout prevents latency reactivation in a primary CD4+ T cell model of HIV latency without affecting activation of these cells. RNA sequencing data showed that CCNT1 regulates HIV-1 proviral genes to a larger extent than any other host gene and had no significant effects on RNA transcripts in primary T cells after activation. We conclude that CCNT1 function is redundant in T cells but is absolutely required for HIV latency reversal.

Primate TRIM34 is a broadly-acting, TRIM5-dependent lentiviral restriction factor.

Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIVAGM-SAB, SIVAGM-TAN and SIVMAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. However, in all cases, this restriction also required the presence of TRIM5α. We demonstrate that TRIM5α is necessary, but not sufficient, for restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY v1 loop and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data support a model in which TRIM34 is a broadly-conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone.

A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection.

Inflammasomes are cytosolic innate immune complexes that assemble upon detection of diverse pathogen-associated cues and play a critical role in host defense and inflammatory pathogenesis. Here, we find that the human inflammasome-forming sensor CARD8 senses HIV-1 infection via site-specific cleavage of the CARD8 N-terminus by the HIV protease (HIV-1PR). HIV-1PR cleavage of CARD8 induces pyroptotic cell death and the release of pro-inflammatory cytokines from infected cells, processes regulated by Toll-like receptor stimulation prior to viral infection. In acutely infected cells, CARD8 senses the activity of both de novo translated HIV-1PR and packaged HIV-1PR that is released from the incoming virion. Moreover, our evolutionary analyses reveal that the HIV-1PR cleavage site in human CARD8 arose after the divergence of chimpanzees and humans. Although chimpanzee CARD8 does not recognize proteases from HIV or simian immunodeficiency viruses from chimpanzees (SIVcpz), SIVcpz does cleave human CARD8, suggesting that SIVcpz was poised to activate the human CARD8 inflammasome prior to its cross-species transmission into humans. Our findings suggest a unique role for CARD8 inflammasome activation in response to lentiviral infection of humans.

Primate TRIM34 is a broadly-acting, TRIM5-dependent lentiviral restriction factor.

Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIV AGM-SAB , SIV AGM-TAN and SIV MAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. However, in all cases, this restriction also required the presence of TRIM5α. We demonstrate that TRIM5α is necessary, but not sufficient, for restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY v1 loop and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data support a model in which TRIM34 is a broadly-conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone.

A Virus-Packageable CRISPR System Identifies Host Dependency Factors Co-Opted by Multiple HIV-1 Strains.

At each stage of the HIV life cycle, host cellular proteins are hijacked by the virus to establish and enhance infection. We adapted the virus packageable HIV-CRISPR screening technology at a genome-wide scale to comprehensively identify host factors that affect HIV replication in a human T cell line. Using a smaller, targeted HIV Dependency Factor (HIVDEP) sublibrary, we then performed screens across HIV strains representing different clades and with different biological properties to define which T cell host factors are important across multiple HIV strains. Nearly 90% of the genes selected across various host pathways validated in subsequent assays as bona fide host dependency factors, including numerous proteins not previously reported to play roles in HIV biology, such as UBE2M, MBNL1, FBXW7, PELP1, SLC39A7, and others. Our ranked list of screen hits across diverse HIV-1 strains form a resource of HIV dependency factors for future investigation of host proteins involved in HIV biology. IMPORTANCE With a small genome of ~9.2 kb that encodes 14 major proteins, HIV must hijack host cellular machinery to successfully establish infection. These host proteins necessary for HIV replication are called "dependency factors." Whole-genome, and then targeted screens were done to try to comprehensively identify all dependency factors acting throughout the HIV replication cycle. Many host processes were identified and validated as critical for HIV replication across multiple HIV strains.

The structural basis for HIV-1 Vif antagonism of human APOBEC3G.

The APOBEC3 (A3) proteins are host antiviral cellular proteins that hypermutate the viral genome of diverse viral families. In retroviruses, this process requires A3 packaging into viral particles1-4. The lentiviruses encode a protein, Vif, that antagonizes A3 family members by targeting them for degradation. Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses. How this 'molecular arms race' plays out at the structural level is unknown. Here, we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif, and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis. A small surface explains the molecular arms race, including a cross-species transmission event that led to the birth of HIV-1. Unexpectedly, we find that RNA is a molecular glue for the Vif-A3G interaction, enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms. Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus-when bound to RNA and on the pathway to packaging-to prevent viral restriction. By engaging essential surfaces required for restriction, Vif exploits a vulnerability in A3G, suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene.

A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency.

Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NFκB pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Evolutionary Landscapes of Host-Virus Arms Races.

Vertebrate immune systems suppress viral infection using both innate restriction factors and adaptive immunity. Viruses mutate to escape these defenses, driving hosts to counterevolve to regain fitness. This cycle recurs repeatedly, resulting in an evolutionary arms race whose outcome depends on the pace and likelihood of adaptation by host and viral genes. Although viruses evolve faster than their vertebrate hosts, their proteins are subject to numerous functional constraints that impact the probability of adaptation. These constraints are globally defined by evolutionary landscapes, which describe the fitness and adaptive potential of all possible mutations. We review deep mutational scanning experiments mapping the evolutionary landscapes of both host and viral proteins engaged in arms races. For restriction factors and some broadly neutralizing antibodies, landscapes favor the host, which may help to level the evolutionary playing field against rapidly evolving viruses. We discuss the biophysical underpinnings of these landscapes and their therapeutic implications.