Using structure prediction of negative sense RNA virus nucleoproteins to assess evolutionary relationships.

Negative sense RNA viruses (NSV) include some of the most detrimental human pathogens, including the influenza, Ebola, and measles viruses. NSV genomes consist of one or multiple single-stranded RNA molecules that are encapsidated into one or more ribonucleoprotein (RNP) complexes. These RNPs consist of viral RNA, a viral RNA polymerase, and many copies of the viral nucleoprotein (NP). Current evolutionary relationships within the NSV phylum are based on the alignment of conserved RNA-dependent RNA polymerase (RdRp) domain amino acid sequences. However, the RdRp domain-based phylogeny does not address whether NP, the other core protein in the NSV genome, evolved along the same trajectory or whether several RdRp-NP pairs evolved through convergent evolution in the segmented and non-segmented NSV genome architectures. Addressing how NP and the RdRp domain evolved may help us better understand NSV diversity. Since NP sequences are too short to infer robust phylogenetic relationships, we here used experimentally obtained and AlphaFold 2.0-predicted NP structures to probe whether evolutionary relationships can be estimated using NSV NP sequences. Following flexible structure alignments of modeled structures, we find that the structural homology of the NSV NPs reveals phylogenetic clusters that are consistent with RdRp-based clustering. In addition, we were able to assign viruses for which RdRp sequences are currently missing to phylogenetic clusters based on the available NP sequence. Both our RdRp-based and NP-based relationships deviate from the current NSV classification of the segmented Naedrevirales, which cluster with the other segmented NSVs in our analysis. Overall, our results suggest that the NSV RdRp and NP genes largely evolved along similar trajectories and even short pieces of genetic, protein-coding information can be used to infer evolutionary relationships, potentially making metagenomic analyses more valuable.

RNA Polymerase Inhibitor Enisamium for Treatment of Moderate COVID-19 Patients: A Randomized, Placebo-Controlled, Multicenter, Double-Blind Phase 3 Clinical Trial.

Enisamium is an orally available therapeutic that inhibits influenza A virus and SARS-CoV-2 replication. We evaluated the clinical efficacy of enisamium treatment combined with standard care in adult, hospitalized patients with moderate COVID-19 requiring external oxygen. Hospitalized patients with laboratory-confirmed SARS-CoV-2 infection were randomly assigned to receive either enisamium (500 mg per dose, four times a day) or a placebo. The primary outcome was an improvement of at least two points on an eight-point severity rating (SR) scale within 29 days of randomization. We initially set out to study the effect of enisamium on patients with a baseline SR of 4 or 5. However, because the study was started early in the COVID-19 pandemic, and COVID-19 had been insufficiently studied at the start of our study, an interim analysis was performed alongside a conditional power analysis in order to ensure patient safety and assess whether the treatment was likely to be beneficial for one or both groups. Following this analysis, a beneficial effect was observed for patients with an SR of 4 only, i.e., patients with moderate COVID-19 requiring supplementary oxygen. The study was continued for these COVID-19 patients. Overall, a total of 592 patients were enrolled and randomized between May 2020 and March 2021. Patients with a baseline SR of 4 were divided into two groups: 142 (49.8%) were assigned to the enisamium group and 143 (50.2%) to the placebo group. An analysis of the population showed that if patients were treated within 4 days of the onset of COVID-19 symptoms (n = 33), the median time to improvement was 8 days for the enisamium group and 13 days for the placebo group (p = 0.005). For patients treated within 10 days of the onset of COVID-19 symptoms (n = 154), the median time to improvement was 10 days for the enisamium group and 12 days for the placebo group (p = 0.002). Our findings suggest that enisamium is safe to use with COVID-19 patients, and that the observed clinical benefit of enisamium is worth reporting and studying in detail.

Long-term evolution of human seasonal influenza virus A(H3N2) is associated with an increase in polymerase complex activity.

Since the influenza pandemic in 1968, influenza A(H3N2) viruses have become endemic. In this state, H3N2 viruses continuously evolve to overcome immune pressure as a result of prior infection or vaccination, as is evident from the accumulation of mutations in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, phylogenetic studies have also demonstrated ongoing evolution in the influenza A(H3N2) virus RNA polymerase complex genes. The RNA polymerase complex of seasonal influenza A(H3N2) viruses produces mRNA for viral protein synthesis and replicates the negative sense viral RNA genome (vRNA) through a positive sense complementary RNA intermediate (cRNA). Presently, the consequences and selection pressures driving the evolution of the polymerase complex remain largely unknown. Here, we characterize the RNA polymerase complex of seasonal influenza A(H3N2) viruses representative of nearly 50 years of influenza A(H3N2) virus evolution. The H3N2 polymerase complex is a reassortment of human and avian influenza virus genes. We show that since 1968, influenza A(H3N2) viruses have increased the transcriptional activity of the polymerase complex while retaining a close balance between mRNA, vRNA, and cRNA levels. Interestingly, the increased polymerase complex activity did not result in increased replicative ability on differentiated human airway epithelial (HAE) cells. We hypothesize that the evolutionary increase in polymerase complex activity of influenza A(H3N2) viruses may compensate for the reduced HA receptor binding and avidity that is the result of the antigenic evolution of influenza A(H3N2) viruses.

Using structure prediction of negative sense RNA virus nucleoproteins to assess evolutionary relationships.

Negative sense RNA viruses (NSV) include some of the most detrimental human pathogens, including the influenza, Ebola and measles viruses. NSV genomes consist of one or multiple single-stranded RNA molecules that are encapsidated into one or more ribonucleoprotein (RNP) complexes. These RNPs consist of viral RNA, a viral RNA polymerase, and many copies of the viral nucleoprotein (NP). Current evolutionary relationships within the NSV phylum are based on alignment of conserved RNA-directed RNA polymerase (RdRp) domain amino acid sequences. However, the RdRp domain-based phylogeny does not address whether NP, the other core protein in the NSV genome, evolved along the same trajectory or whether several RdRp-NP pairs evolved through convergent evolution in the segmented and non-segmented NSV genomes architectures. Addressing how NP and the RdRp domain evolved may help us better understand NSV diversity. Since NP sequences are too short to infer robust phylogenetic relationships, we here used experimentally-obtained and AlphaFold 2.0-predicted NP structures to probe whether evolutionary relationships can be estimated using NSV NP sequences. Following flexible structure alignments of modeled structures, we find that the structural homology of the NSV NPs reveals phylogenetic clusters that are consistent with RdRp-based clustering. In addition, we were able to assign viruses for which RdRp sequences are currently missing to phylogenetic clusters based on the available NP sequence. Both our RdRp-based and NP-based relationships deviate from the current NSV classification of the segmented Naedrevirales, which cluster with the other segmented NSVs in our analysis. Overall, our results suggest that the NSV RdRp and NP genes largely evolved along similar trajectories and that even short pieces of genetic, protein-coding information can be used to infer evolutionary relationships, potentially making metagenomic analyses more valuable.

Transient RNA structures underlie highly pathogenic avian influenza virus genesis.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease and high fatality in poultry1. They emerge exclusively from H5 and H7 low pathogenic avian influenza viruses (LPAIVs)2. Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for LPAIV-to-HPAIV transition3,4, the exact mechanisms underlying said insertion have remained unknown. Here we used an innovative combination of bioinformatic models to predict RNA structures forming around the influenza virus RNA polymerase during replication, and circular sequencing5 to reliably detect nucleotide insertions. We show that transient H5 hemagglutinin RNA structures predicted to trap the polymerase on purine-rich sequences drive nucleotide insertions characteristic of MBCSs, providing the first strong empirical evidence of RNA structure involvement in MBCS acquisition. Insertion frequencies at the H5 cleavage site were strongly affected by substitutions in flanking genomic regions altering predicted transient RNA structures. Introduction of H5-like cleavage site sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions never observed before in H6 viruses. Our results demonstrate that nucleotide insertions that underlie H5 HPAIV emergence result from a previously unknown RNA-structure-driven diversity-generating mechanism, which could be shared with other RNA viruses.

RNA structure modulates Cas13 activity and enables mismatch detection.

The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging1-6. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA1,7. Though Cas13 is not activated by double-stranded RNA (dsRNA) in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured2,8. Understanding Cas13's mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown9. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3' to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.

Cas13-based amplification-free quantification of aberrant RNAs during influenza virus infection.

Influenza A virus RNA synthesis produces full-length and aberrant RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that several hundred unique aberrant RNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Understanding the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for modeling the outcomes of IAV infection. We here first show that reverse transcription and PCR steps can yield imperfect aberrant RNA quantification data in a sequence-dependent manner. Next, we developed an amplification-free LbuCas13a-based detection method to quantify mvRNA amplification kinetics and subcellular distributions. We show that our assay can quantify the copy numbers of 10 specific mvRNA sequences in total RNA from cell culture, animal tissue or clinical nasopharyngeal swab extracts. In addition, we find kinetic and distribution differences between immunostimulatory and non-immunostimulatory mvRNAs, as well as mvRNAs derived from different segments, during infection. Overall, our results reveal a hitherto hidden diversity in the behavior of IAV mvRNAs and they suggest that their production is linked to replication of the individual viral segments. Cas13 is therefore a valuable new tool in our repertoire for investigating the impact of aberrant RNAs on RNA virus infection.

Deep mutational scanning reveals the functional constraints and evolutionary potential of the influenza A virus PB1 protein.

IMPORTANCE: The influenza virus polymerase is important for adaptation to new hosts and, as a determinant of mutation rate, for the process of adaptation itself. We performed a deep mutational scan of the polymerase basic 1 (PB1) protein to gain insights into the structural and functional constraints on the influenza RNA-dependent RNA polymerase. We find that PB1 is highly constrained at specific sites that are only moderately predicted by the global structure or larger domain. We identified a number of beneficial mutations, many of which have been shown to be functionally important or observed in influenza virus' natural evolution. Overall, our atlas of PB1 mutations and their fitness impacts serves as an important resource for future studies of influenza replication and evolution.

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.

Negative and ambisense RNA virus ribonucleocapsids: more than protective armor.

SUMMARYNegative and ambisense RNA viruses are the causative agents of important human diseases such as influenza, measles, Lassa fever, and Ebola hemorrhagic fever. The viral genome of these RNA viruses consists of one or more single-stranded RNA molecules that are encapsidated by viral nucleocapsid proteins to form a ribonucleoprotein complex (RNP). This RNP acts as protection, as a scaffold for RNA folding, and as the context for viral replication and transcription by a viral RNA polymerase. However, the roles of the viral nucleoproteins extend beyond these functions during the viral infection cycle. Recent advances in structural biology techniques and analysis methods have provided new insights into the formation, function, dynamics, and evolution of negative sense virus nucleocapsid proteins, as well as the role that they play in host innate immune responses against viral infection. In this review, we discuss the various roles of nucleocapsid proteins, both in the context of RNPs and in RNA-free states, as well as the open questions that remain.

Relative role of community transmission and campus contagion in driving the spread of SARS-CoV-2: Lessons from Princeton University.

Mathematical models have played a crucial role in exploring and guiding pandemic responses. University campuses present a particularly well-documented case for institutional outbreaks, thereby providing a unique opportunity to understand detailed patterns of pathogen spread. Here, we present descriptive and modeling analyses of SARS-CoV-2 transmission on the Princeton University (PU) campus-this model was used throughout the pandemic to inform policy decisions and operational guidelines for the university campus. Epidemic patterns between the university campus and surrounding communities exhibit strong spatiotemporal correlations. Mathematical modeling analysis further suggests that the amount of on-campus transmission was likely limited during much of the wider pandemic until the end of 2021. Finally, we find that a superspreading event likely played a major role in driving the Omicron variant outbreak on the PU campus during the spring semester of the 2021-2022 academic year. Despite large numbers of cases on campus in this period, case levels in surrounding communities remained low, suggesting that there was little spillover transmission from campus to the local community.

Mutual information networks reveal evolutionary relationships within the influenza A virus polymerase.

The influenza A virus (IAV) RNA polymerase is an essential driver of IAV evolution. Mutations that the polymerase introduces into viral genome segments during replication are the ultimate source of genetic variation, including within the three subunits of the IAV polymerase (polymerase basic protein 2, polymerase basic protein 1, and polymerase acidic protein). Evolutionary analysis of the IAV polymerase is complicated, because changes in mutation rate, replication speed, and drug resistance involve epistatic interactions among its subunits. In order to study the evolution of the human seasonal H3N2 polymerase since the 1968 pandemic, we identified pairwise evolutionary relationships among ∼7000 H3N2 polymerase sequences using mutual information (MI), which measures the information gained about the identity of one residue when a second residue is known. To account for uneven sampling of viral sequences over time, we developed a weighted MI (wMI) metric and demonstrate that wMI outperforms raw MI through simulations using a well-sampled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dataset. We then constructed wMI networks of the H3N2 polymerase to extend the inherently pairwise wMI statistic to encompass relationships among larger groups of residues. We included hemagglutinin (HA) in the wMI network to distinguish between functional wMI relationships within the polymerase and those potentially due to hitch-hiking on antigenic changes in HA. The wMI networks reveal coevolutionary relationships among residues with roles in replication and encapsidation. Inclusion of HA highlighted polymerase-only subgraphs containing residues with roles in the enzymatic functions of the polymerase and host adaptability. This work provides insight into the factors that drive and constrain the rapid evolution of influenza viruses.

Generation of host-directed and virus-specific antivirals using targeted protein degradation promoted by small molecules and viral RNA mimics.

Targeted protein degradation (TPD), as exemplified by proteolysis-targeting chimera (PROTAC), is an emerging drug discovery platform. PROTAC molecules, which typically contain a target protein ligand linked to an E3 ligase ligand, recruit a target protein to the E3 ligase to induce its ubiquitination and degradation. Here, we applied PROTAC approaches to develop broad-spectrum antivirals targeting key host factors for many viruses and virus-specific antivirals targeting unique viral proteins. For host-directed antivirals, we identified a small-molecule degrader, FM-74-103, that elicits selective degradation of human GSPT1, a translation termination factor. FM-74-103-mediated GSPT1 degradation inhibits both RNA and DNA viruses. Among virus-specific antivirals, we developed viral RNA oligonucleotide-based bifunctional molecules (Destroyers). As a proof of principle, RNA mimics of viral promoter sequences were used as heterobifunctional molecules to recruit and target influenza viral polymerase for degradation. This work highlights the broad utility of TPD to rationally design and develop next-generation antivirals.

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consists of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the IBV genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.

Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets.

Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.

Mutual information networks reveal evolutionary relationships within the influenza A virus polymerase.

The influenza A (IAV) RNA polymerase is an essential driver of IAV evolution. Mutations that the polymerase introduces into viral genome segments during replication are the ultimate source of genetic variation, including within the three subunits of the IAV polymerase (PB2, PB1, and PA). Evolutionary analysis of the IAV polymerase is complicated, because changes in mutation rate, replication speed, and drug resistance involve epistatic interactions among its subunits. In order to study the evolution of the human seasonal H3N2 polymerase since the 1968 pandemic, we identified pairwise evolutionary relationships among ∻7000 H3N2 polymerase sequences using mutual information (MI), which measures the information gained about the identity of one residue when a second residue is known. To account for uneven sampling of viral sequences over time, we developed a weighted MI metric (wMI) and demonstrate that wMI outperforms raw MI through simulations using a well-sampled SARS-CoV-2 dataset. We then constructed wMI networks of the H3N2 polymerase to extend the inherently pairwise wMI statistic to encompass relationships among larger groups of residues. We included HA in the wMI network to distinguish between functional wMI relationships within the polymerase and those potentially due to hitchhiking on antigenic changes in HA. The wMI networks reveal coevolutionary relationships among residues with roles in replication and encapsidation. Inclusion of HA highlighted polymerase-only subgraphs containing residues with roles in the enzymatic functions of the polymerase and host adaptability. This work provides insight into the factors that drive and constrain the rapid evolution of influenza viruses.

Transient RNA structures cause aberrant influenza virus replication and innate immune activation.

During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid-inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.

Decoding the Role of Temperature in RNA Virus Infections.

RNA viruses include respiratory viruses, such as coronaviruses and influenza viruses, as well as vector-borne viruses, like dengue and West Nile virus. RNA viruses like these encounter various environments when they copy themselves and spread from cell to cell or host to host. Ex vivo differences, such as geographical location and humidity, affect their stability and transmission, while in vivo differences, such as pH and host gene expression, impact viral receptor binding, viral replication, and the host immune response against the viral infection. A critical factor affecting RNA viruses both ex vivo and in vivo, and defining the outcome of viral infections and the direction of viral evolution, is temperature. In this minireview, we discuss the impact of temperature on viral replication, stability, transmission, and adaptation, as well as the host innate immune response. Improving our understanding of how RNA viruses function, survive, and spread at different temperatures will improve our models of viral replication and transmission risk analyses.

Tinker, tailor, antiviral: RNA virus inhibition by induced recombination.

Nucleotide analogs can help to combat RNA virus growth by stalling the viral RNA polymerase or by introducing lethal mutations into the viral genome. Janissen and Woodman et al. have used single-molecule, sequencing, and virological methods to reveal that antiviral T-1106 provides a third mechanism of counterattack: inducing recombination.

The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis.

Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is wound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the cRNA, and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases. Differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from cRNA but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32A is not only needed for the actively replicating polymerase, but not for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation. IMPORTANCE Zoonotic avian influenza A viruses pose a constant threat to global health, and they have the potential to cause pandemics. Species variations in host factor ANP32A play a key role in supporting the activity of avian influenza A virus RNA polymerases in mammalian hosts. Here we show that ANP32A acts at two stages in the influenza A virus replication cycle, supporting recent structural experiments, in line with its essential role. Understanding how ANP32A supports viral RNA polymerase activity and how it supports avian polymerase function in mammalian hosts is important for understanding influenza A virus replication and the development of antiviral strategies against influenza A viruses.