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.

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 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.

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.

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.

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.

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.

HIV-1 Vif Gained Breadth in APOBEC3G Specificity after Cross-Species Transmission of Its Precursors.

APOBEC3G (A3G) is a host-encoded cytidine deaminase that potently restricts retroviruses such as HIV-1 and depends on its ability to package into virions. As a consequence of this, HIV-1 protein Vif has evolved to antagonize human A3G by targeting it for ubiquitination and subsequent degradation. There is an ancient arms race between Vif and A3G highlighted by amino acids 128 and 130 in A3G that have evolved under positive selection due to Vif-mediated selective pressure in Old World primates. Nonetheless, not all possible amino acid combinations at these sites have been sampled by nature, and the evolutionary potential of species to resist Vif antagonism is not clear. To explore the evolutionary space of positively selected sites in the Vif-binding region of A3G, we designed a combinatorial mutagenesis screen to introduce all 20 amino acids at sites 128 and 130. Our screen uncovered mutants of A3G with several interesting phenotypes, including loss of antiviral activity and resistance of Vif antagonism. However, HIV-1 Vif exhibited remarkable flexibility in antagonizing A3G 128 and 130 mutants, which significantly reduces viable Vif resistance strategies for hominid primates. Importantly, we find that broadened Vif specificity was conferred through loop 5 adaptations that were required for cross-species adaptation from Old World monkey A3G to hominid A3G. Our evidence suggests that Vif adaptation to novel A3G interfaces during cross-species transmission may train Vif toward broadened specificity that can further facilitate cross-species transmissions and raise the barrier to host resistance. IMPORTANCE APOBEC3G (A3G) is an antiviral protein that potently restricts retroviruses like HIV. In turn, the HIV-1 protein Vif has evolved to antagonize A3G through degradation. Two rapidly evolving sites in A3G confer resistance to unadapted Vif and act as a barrier to cross-species transmission of retroviruses. We recently identified a single amino acid mutation in a simian immunodeficiency virus (SIV) Vif that contributed to the cross-species origins of SIV infecting chimpanzee and, ultimately, the HIV-1 pandemic. This mutation broadened specificity of this Vif to both antagonize the A3G of its host while simultaneously overcoming the A3G barrier in the great apes. In this work, we explore the evolutionary space of human A3G at these rapidly evolving sites to understand if the broadened Vif specificity gained during cross-species transmission confers an advantage to HIV-1 Vif in its host-virus arms race with A3G.

Divergence in Dimerization and Activity of Primate APOBEC3C.

The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and we determined that Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity, 44, 45, and 144, also promoted dimerization and changes to the dynamics of loop 1, near the enzyme active site. Although forced evolution of rhA3C resulted in a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C and establish the amino acid network for dimerization and increased activity. Based on identification of the key amino acids determining Old World Monkey antiviral activity we predict that other Old World Monkey A3Cs did not impart anti-lentiviral activity, despite fixation of a key residue needed for hominid A3C activity. Overall, the coevolutionary analysis of the A3C dimerization interface presented also provides a basis from which to analyze dimerization interfaces of other A3 family members.

Highly-potent, synthetic APOBEC3s restrict HIV-1 through deamination-independent mechanisms.

The APOBEC3 (A3) genes encode cytidine deaminase proteins with potent antiviral and anti-retroelement activity. This locus is characterized by duplication, recombination, and deletion events that gave rise to the seven A3s found in primates. These include three single deaminase domain A3s (A3A, A3C, and A3H) and four double deaminase domain A3s (A3B, A3D, A3F, and A3G). The most potent of the A3 proteins against HIV-1 is A3G. However, it is not clear if double deaminase domain A3s have a generalized functional advantage to restrict HIV-1. In order to test whether superior restriction factors could be created by genetically linking single A3 domains into synthetic double domains, we linked A3C and A3H single domains in novel combinations. We found that A3C/A3H double domains acquired enhanced antiviral activity that is at least as potent, if not better than, A3G. Although these synthetic double domain A3s package into budding virions more efficiently than their respective single domains, this does not fully explain their gain of antiviral potency. The antiviral activity is conferred both by cytidine-deaminase dependent and independent mechanisms, with the latter correlating to an increase in RNA binding affinity. T cell lines expressing this A3C-A3H super restriction factor are able to control replicating HIV-1ΔVif infection to similar levels as A3G. Together, these data show that novel combinations of A3 domains are capable of gaining potent antiviral activity to levels similar to the most potent genome-encoded A3s, via a primarily non-catalytic mechanism.

Mutational resilience of antiviral restriction favors primate TRIM5α in host-virus evolutionary arms races.

Host antiviral proteins engage in evolutionary arms races with viruses, in which both sides rapidly evolve at interaction interfaces to gain or evade immune defense. For example, primate TRIM5α uses its rapidly evolving 'v1' loop to bind retroviral capsids, and single mutations in this loop can dramatically improve retroviral restriction. However, it is unknown whether such gains of viral restriction are rare, or if they incur loss of pre-existing function against other viruses. Using deep mutational scanning, we comprehensively measured how single mutations in the TRIM5α v1 loop affect restriction of divergent retroviruses. Unexpectedly, we found that the majority of mutations increase weak antiviral function. Moreover, most random mutations do not disrupt potent viral restriction, even when it is newly acquired via a single adaptive substitution. Our results indicate that TRIM5α's adaptive landscape is remarkably broad and mutationally resilient, maximizing its chances of success in evolutionary arms races with retroviruses.

The evolutionary battle between viruses and the immune system is essentially a high-stakes arms race. The immune system makes antiviral proteins, called restriction factors, which can stop the virus from replicating. In response, viruses evolve to evade the effects of restriction factors. To counter this, restriction factors evolve too, and the cycle continues. The challenge for the immune system is that mammals do not evolve as fast as viruses. How then, in the face of this disadvantage, can the immune system hope to keep pace with viral evolution? One human antiviral protein that seems to have struggled to keep up is TRIM5α. In rhesus macaques, it is very effective at stopping the replication of HIV-1 and related viruses. But in humans, it is not effective at all. But why? Protein evolution happens due to small genetic mutations, but not every mutation makes a protein better. If a protein is resilient, it can tolerate lots of neutral or negative mutations without breaking, until it mutates in a way that makes it better. But, if a protein is fragile, even small changes can render it completely unable to do its job. It is possible that restriction factors, like TRIM5α, are evolutionarily 'fragile', and therefore easy to break. But it is difficult to test whether this is the case, because existing mutations have already passed the test of natural selection. This means that either the mutation is somehow useful for the protein, or that it is not harmful enough to be removed. Tenthorey et al. devised a way to introduce all possible changes to the part of TRIM5α that binds to viruses. This revealed that TRIM5α is not fragile; most random mutations increased, rather than decreased, the protein's ability to prevent viral infection. In fact, it appears it would only take a single mutation to make TRIM5α better at blocking HIV-1 in humans, and there are many possible single mutations that would work. Thus, it would appear that human TRIM5α can easily gain the ability to block HIV-1. The next step was to find out whether these gains in antiviral activity are just as easily lost. To do this, Tenthorey et al. performed the same tests on TRIM5α from rhesus macaques and an HIV-blocking mutant version of human TRIM5α. This showed that the majority of random mutations do not break TRIM5α's virus-blocking ability. Thus, TRIM5α can readily gain antiviral activity and, once gained, does not lose it easily during subsequent mutation. Antiviral proteins like TRIM5α engage in uneven evolutionary battles with fast-evolving viruses. But, although they are resilient and able to evolve, they are not always able to find the right mutations on their own. Experiments like these suggest that it might be possible to give them a helping hand. Identifying mutations that help human TRIM5α to strongly block HIV-1 could pave the way for future gene therapy. This step would demand significant advances in gene therapy efficacy and safety, but it could offer a new way to block virus infection in the future.