The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction.

Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans-Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription.IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency.

Using SHAPE-MaP To Model RNA Secondary Structure and Identify 3'UTR Variation in Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3'UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3'UTR variants. A duplication in the 3'UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3'UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3'UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.

mTORC2 Activity Disrupts Lysosome Acidification in Systemic Lupus Erythematosus by Impairing Caspase-1 Cleavage of Rab39a.

Lysosomes maintain immune homeostasis through the degradation of phagocytosed apoptotic debris; however, the signaling events regulating lysosomal maturation remain undefined. In this study, we show that lysosome acidification, key to the maturation process, relies on mTOR complex 2 (mTORC2), activation of caspase-1, and cleavage of Rab39a. Mechanistically, the localization of cofilin to the phagosome recruits caspase-11, which results in the localized activation of caspase-1. Caspase-1 subsequently cleaves Rab39a on the phagosomal membrane, promoting lysosome acidification. Although caspase-1 is critical for lysosome acidification, its activation is independent of inflammasomes and cell death mediated by apoptosis-associated speck-like protein containing a caspase recruitment domain, revealing a role beyond pyroptosis. In lupus-prone murine macrophages, chronic mTORC2 activity decouples the signaling pathway, leaving Rab39a intact. As a result, the lysosome does not acidify, and degradation is impaired, thereby heightening the burden of immune complexes that activate FcγRI and sustain mTORC2 activity. This feedforward loop promotes chronic immune activation, leading to multiple lupus-associated pathologies. In summary, these findings identify the key molecules in a previously unappreciated signaling pathway that promote lysosome acidification. It also shows that this pathway is disrupted in systemic lupus erythematosus.

Structural divergence creates new functional features in alphavirus genomes.

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins.

An RNA structure-mediated, posttranscriptional model of human α-1-antitrypsin expression.

Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5'-untranslated region (5'-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5'-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2'-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5'-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5'-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.

Inducing circular RNA formation using the CRISPR endoribonuclease Csy4.

Circular RNAs (circRNAs) are highly stable, covalently closed RNAs that are regulated in a spatiotemporal manner and whose functions are largely unknown. These molecules have the potential to be incorporated into engineered systems with broad technological implications. Here we describe a switch for inducing back-splicing of an engineered circRNA that relies on the CRISPR endoribonuclease, Csy4, as an activator of circularization. The endoribonuclease activity and 3' end-stabilizing properties of Csy4 are particularly suited for this task. Coexpression of Csy4 and the circRNA switch allows for the removal of downstream competitive splice sites and stabilization of the 5' cleavage product. This subsequently results in back-splicing of the 5' cleavage product into a circRNA that can translate a reporter protein from an internal ribosomal entry site (IRES). Our platform outlines a straightforward approach toward regulating splicing and could find potential applications in synthetic biology as well as in studying the properties of different circRNAs.

Kinome Profiling Identifies Druggable Targets for Novel Human Cytomegalovirus (HCMV) Antivirals.

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of <1 µm, and at doses that were not toxic to uninfected cells. The most potent inhibitor of HCMV replication was OTSSP167 (IC50 <1.2 nm), a MELK inhibitor, blocked HCMV early gene expression and viral DNA accumulation, resulting in a >3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1.

Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2α. Consistent with previous in vitro studies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2α binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2α contact site in PKR binding, pTRS1 bound an additional eIF2α kinase, heme-regulated inhibitor (HRI), and inhibited eIF2α phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2α binding site and blocking PKR kinase activity.IMPORTANCE The antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease.

Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication.

UNLABELLED: Human cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication. IMPORTANCE: To successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease.

Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection.

UNLABELLED: Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5' untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection. IMPORTANCE: The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.

Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms.

The membrane associated accessory protein is an adeno-associated viral egress factor.

Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

Engineering highly efficient backsplicing and translation of synthetic circRNAs.

Circular RNAs (circRNAs) are highly stable RNA molecules that are attractive templates for expression of therapeutic proteins and non-coding RNAs. In eukaryotes, circRNAs are primarily generated by the spliceosome through backsplicing. Here, we interrogate different molecular elements including intron type and length, Alu repeats, internal ribosome entry sites (IRESs), and exon length essential for circRNA formation and exploit this information to engineer robust backsplicing and circRNA expression. Specifically, we leverage the finding that the downstream intron can tolerate large inserts without affecting splicing to achieve tandem expression of backspliced circRNAs and tRNA intronic circRNAs from the same template. Further, truncation of selected intronic regions markedly increased circRNA formation in different cell types in vitro as well as AAV-mediated circRNA expression in cardiac and skeletal muscle tissue in vivo. We also observed that different IRES elements and exon length influenced circRNA expression and translation, revealing an exonic contribution to splicing, as evidenced by different RNA species produced. Taken together, these data provide new insight into improving the design and expression of synthetic circRNAs. When combined with AAV capsid and promoter technologies, the backsplicing introns and IRES elements constituting this modular platform significantly expand the gene expression toolkit.

Comparative analysis of coronavirus genomic RNA structure reveals conservation in SARS-like coronaviruses.

Coronaviruses, including SARS-CoV-2 the etiological agent of COVID-19 disease, have caused multiple epidemic and pandemic outbreaks in the past 20 years1-3. With no vaccines, and only recently developed antiviral therapeutics, we are ill equipped to handle coronavirus outbreaks4. A better understanding of the molecular mechanisms that regulate coronavirus replication and pathogenesis is needed to guide the development of new antiviral therapeutics and vaccines. RNA secondary structures play critical roles in multiple aspects of coronavirus replication, but the extent and conservation of RNA secondary structure across coronavirus genomes is unknown5. Here, we define highly structured RNA regions throughout the MERS-CoV, SARS-CoV, and SARS-CoV-2 genomes. We find that highly stable RNA structures are pervasive throughout coronavirus genomes, and are conserved between the SARS-like CoV. Our data suggests that selective pressure helps preserve RNA secondary structure in coronavirus genomes, suggesting that these structures may play important roles in virus replication and pathogenesis. Thus, disruption of conserved RNA secondary structures could be a novel strategy for the generation of attenuated SARS-CoV-2 vaccines for use against the current COVID-19 pandemic.

Human cytomegalovirus pTRS1 stimulates cap-independent translation.

Human cytomegalovirus (HCMV) manipulates multiple cellular processes to facilitate virus replication, including the control of mRNA translation. We previously showed that the HCMV TRS1 protein (pTRS1) promotes cap-dependent mRNA translation independent of its ability to antagonize the antiviral protein PKR. Here we find that pTRS1 enhances internal ribosome entry site (IRES) activity using a novel circular RNA reporter that lacks an mRNA cap and poly(A) tail. Additionally, pTRS1 expression increases the activity of cellular IRESs that control the expression of proteins needed for efficient HCMV replication. We find that the ability of pTRS1 to enhance cap-independent translation is separable from its ability to antagonize PKR, but requires the pTRS1 RNA binding domain. Together these data show that pTRS1 stimulates cap-independent translation and suggest a role for pTRS1 in alternative translation initiation pathways during HCMV infection.

Precision mouse models with expanded tropism for human pathogens.

A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics.

Complete Genome Sequences of 15 Chikungunya Virus Isolates from Puerto Rico.

Here, we report the complete genome sequences of 15 chikungunya virus strains isolated from human plasma from infected patients in Puerto Rico. The results show that currently circulating chikungunya strains in Puerto Rico are closely related.

Human Cytomegalovirus Strategies to Maintain and Promote mRNA Translation.

mRNA translation requires the ordered assembly of translation initiation factors and ribosomal subunits on a transcript. Host signaling pathways regulate each step in this process to match levels of protein synthesis to environmental cues. In response to infection, cells activate multiple defenses that limit viral protein synthesis, which viruses must counteract to successfully replicate. Human cytomegalovirus (HCMV) inhibits host defenses that limit viral protein expression and manipulates host signaling pathways to promote the expression of both host and viral proteins necessary for virus replication. Here we review key regulatory steps in mRNA translation, and the strategies used by HCMV to maintain protein synthesis in infected cells.