Biology subjects

Carlson, L.-A.

Publications and source records attributed to Carlson, L.-A..

14 recordsLinked to original sources

bioRxiv2024

Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation

Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum (ER) membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we imaged tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing coat and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virions bud. Naturally occurring furin site variants determine whether virions mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

microbiology↗

bioRxiv2024

The flavivirus protein NS4B recruits the cis-Golgi protein ACBD3 to modify ER-Golgi trafficking for virion release

Flavivirus infection involves extensive remodeling of the endoplasmic reticulum (ER), which is key to both the replication of the viral RNA genome as well as the assembly and release of new virions. Yet, little is known about how viral proteins and host factors cooperatively facilitate such a vast transformation of the ER, and how this influences the different steps of the viral life cycle. In this study, we screened for host proteins that interact with the tick-borne encephalitis virus (TBEV) protein NS4B and found that the top candidates were coupled to trafficking between ER exit sites (ERES) and the Golgi. We characterized the role of ACBD3, one of the identified proteins, in flavivirus infection and show that it interacts with NS4B to promote infection across multiple flavivirus species. Using ACBD3 knockout cells, we found that the depletion of ACBD3 inhibited TBEV replication by preventing the trafficking of virions from the cell. We found that ACBD3 promotes flavivirus infection via a different mechanism than its previously described role in picornavirus infection. ACBD3 was enriched at modified ERES-Golgi contact sites to support virus replication. Therefore, we propose that ACBD3 promotes flavivirus replication by modifying the trafficking between the ERES and the Golgi to enable the release of new virions. Author summaryFlaviviruses including dengue virus and tick-borne encephalitis virus are group of viruses that widely affecting the health of human. During infection, flavivirus particles enter host cells and transform the endoplasmic reticulum (ER), which is the main structure for protein synthesis in cells. New flaviviral particles are produced in the transformed ER and then released to the Golgi apparatus, which is the main structure for protein transport in cells. It is unclear how the particles are transported from the ER to Golgi. Here we screened the factors that interact with viral proteins and identified a Golgi protein called ACBD3 as an important factor supporting flavivirus particles release from ER to Golgi. We showed that ACBD3 is recruited by flavivirus to a modified connection between ER and Golgi for viral particles release from the ER. Our work provides new insights into the fine coordination of virus replication and viral particles transport between organelles inside host cells.

cell biology↗

bioRxiv2023

Giardia intestinalis deoxyadenosine kinase has a unique tetrameric structure that enables high substrate affinity and makes the parasite sensitive to deoxyadenosine analogues

Giardia intestinalis is a protozoan parasite causing giardiasis, a severe, sometimes even life-threatening, diarrheal disease. Giardia is one of only a few known organisms that lack de novo synthesis of DNA building blocks, and the parasite is therefore completely dependent on salvaging deoxyribonucleosides from the host. The deoxyribonucleoside kinases (dNKs) needed for this salvage are generally divided into two structurally distinct families, thymidine kinase 1 (TK1)-like dNKs and non-TK1-like dNKs. We have characterized the G. intestinalis deoxyadenosine kinase and found that it, in contrast to previously studied non-TK1-like dNKs, has a tetrameric structure. Deoxyadenosine was the best natural substrate of the enzyme (KM=1.12 M; Vmax=10.3 mol{middle dot}min-1{middle dot}mg-1), whereas the affinities for deoxyguanosine, deoxyinosine and deoxycytidine were 400-2000 times lower. Deoxyadenosine analogues halogenated at the 2- and/or 2 s-positions were also potent substrates, with comparable EC50 values as the main drug used today, metronidazole, but with the advantage of being usable on metronidazole-resistant parasites. Cryo-EM and 2.1 [A] X-ray structures of the enzyme in complex with the product dAMP (and dADP) showed that the tetramer is kept together by extended N- and C-termini that reach across from one canonical dimer to the next in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a 100-fold decreased deoxyribonucleoside substrate affinity. This is the first example of a non-TK1-like dNK that has a higher substrate affinity as the result of a higher oligomeric state. The development of high substrate affinity could be an evolutionary key factor behind the ability of the parasite to survive solely on deoxyribonucleoside salvage. Authors summaryThe human pathogen Giardia intestinalis is one of only a few organisms that lack ribonucleotide reductase and is therefore completely dependent on salvaging deoxyribonucleosides from the host for the supply of DNA building blocks. We have characterized one of the G. intestinalis salvage enzymes, which was named deoxyadenosine kinase based on its substrate specificity. The enzyme also phosphorylated many deoxyadenosine analogues that were equally efficient in preventing parasite growth as the most used drug today, metronidazole, and also usable against metronidazole-resistant parasites. Structural analysis of the enzyme with cryo-EM and X-ray crystallography showed that the enzyme was unique in its family of deoxyribonucleoside kinases by forming a tetramer and mutational analysis showed that tetramerization is a prerequisite for the high substrate affinity of the enzyme. The ability to gain substrate affinity by increasing the number of enzyme subunits could potentially represent an evolutionary pathway that has assisted the parasite to become able to survive entirely on salvage synthesis of DNA building blocks.

biochemistry↗

bioRxiv2023

In vitro reconstitution reveals membrane clustering and double-stranded RNA recruitment by the enteroviral AAA+ ATPase 2C

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2Cs association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divides the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

microbiology↗

bioRxiv2023

Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomes

The Endosomal Sorting Complex Required for Transport (ESCRT) machinery constitutes a multisubunit protein complex that plays an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, encompassing cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair and autophagy. Given the versatile functionality of ESCRTs and the intricate organizational structure of the ESCRT complex, the targeted modulation of distinct ESCRT-mediated membrane deformations for functional dissection poses a considerable challenge. This study presents a pseudo-natural product targeting IST1-CHMP1B within the ESCRT-III complex. This compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting and exosome biogenesis, it rapidly hinders transferrin receptor (TfR) recycling in cells, resulting in the accumulation of transferrin in perinuclear endosomal recycling tubules. Stalled recycling endosomes acquire unconventional LC3 lipidation, establishing a link between non-canonical LC3 lipidation and endosomal recycling.

cell biology↗

bioRxiv2023

Structural basis for lipid transfer by the ATG2A-ATG9A complex

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes. ATG2A and ATG9A play an essential role in autophagy by mediating lipid transfer and re-equilibration between membranes for autophagosome formation. Here we report the cryo-EM structures of human ATG2A-WIPI4 complex at 3.2 [A], and ATG2A-WIPI4-ATG9A complex at 7 [A] resolution. The ATG2A structure is characterized by a central hydrophobic cavity formed by a network of {beta}-strands that facilitates lipid transfer, and highly flexible N- and C-terminal domains. Molecular dynamics simulations of the ATG2A N-terminal domain revealed the mechanism of lipid-extraction from the donor membranes while the ATG2A-ATG9A complex structure provides insights into the later stages of the lipid transfer reaction. ATG9A-ATG2A structural analysis revealed a 1:1 stoichiometry, directly aligning the ATG9A lateral pore with ATG2A lipid transfer cavity, hence allowing for a direct transfer of lipids from ATG2A. The ATG9A trimer can interact with both N- and C-terminal tip of rod-shaped ATG2A. Cryo-electron tomography of ATG2A-liposome binding states shows that ATG2A tethers lipid vesicles at different orientations. In summary, this study provides a molecular basis for the growth of the phagophore membrane, and lends structural insights into spatially coupled lipid transport and re-equilibration during autophagosome formation.

biochemistry↗

bioRxiv2023

Ribosome clustering and surface layer reorganization in the microsporidian host-invasion apparatus.

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and demonstrates that ribosomes are efficiently transported through polar tubes in a spiral-like parallel arrangement.

biophysics↗

bioRxiv2022

The organization of double-stranded RNA in the chikungunya virus replication organelle

Alphaviruses are mosquito-borne, positive-sense single-stranded RNA viruses. Amongst the alphaviruses, chikungunya virus is notable as a large source of human illness, especially in tropical and subtropical regions. When they invade a cell, alphaviruses generate dedicated organelles for viral genome replication, so-called spherules. Spherules form as outward-facing buds at the plasma membrane, and it has recently been shown that the thin membrane neck that connects this membrane bud with the cytoplasm is guarded by a two-megadalton protein complex that contains all the enzymatic functions necessary for RNA replication. The lumen of the spherules contains a single copy of the negative-strand template RNA, present in a duplex with newly synthesized positive-sense RNA. Less is known about the organization of this double-stranded RNA as compared to the protein components of the spherule. Here, we analyzed cryo-electron tomograms of chikungunya virus spherules in terms of the organization of the double-stranded RNA replication intermediate. We find that the double-stranded RNA has a shortened apparent persistence length as compared to unconstrained double-stranded RNA. Around half of the genome is present in either of five conformations identified by subtomogram classification, each representing a relatively straight segment of ~25-32 nm. Finally, the RNA occupies the spherule lumen at a homogeneous density, but has a preferred orientation to be perpendicular to a vector pointing from the membrane neck towards the spherule center. Taken together, this analysis lays another piece of the puzzle of the highly coordinated alphavirus genome replication.

microbiology↗

bioRxiv2022

Architecture of the chikungunya virus replication organelle

Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.

biophysics↗

bioRxiv2021

Whole-brain fluorescence-MRI coregistration for precise anatomical mapping of virus infection

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combined multimodal imaging (cm-nm scale) with single nuclei RNA- sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection was imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection was limited to grey matter of sensory systems in wildtype mice, but extended into white matter, meninges and choroid plexus in Ifnar-/-mice. Cells in wildtype displayed strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+NK cells, whereas in Ifnar-/-, the absence of this response contributed to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.

microbiology↗

bioRxiv2021

Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal {beta}-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.

microbiology↗

bioRxiv2021

In situ structures of membrane-assisted assembly and selective autophagy of enteroviruses

Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

microbiology↗

bioRxiv2020

The structure of enteric human adenovirus 41 - a leading cause of diarrhoea in children

Human adenovirus (HAdV) types F40 and F41 are a prominent cause of diarrhea and diarrhea-associated mortality in young children worldwide. These enteric HAdVs differ strikingly in tissue tropism and pathogenicity from respiratory and ocular adenoviruses, but the structural basis for this divergence has been unknown. Here we present the first structure of an enteric HAdV - HAdV-F41 - determined by cryo-EM to a resolution of 3.8[A]. The structure reveals extensive alterations to the virion exterior as compared to non-enteric HAdVs, including a unique arrangement of capsid protein IX. The structure also provides new insights into conserved aspects of HAdV architecture such as a proposed location of protein V, which links the viral DNA to the capsid, and assembly-induced conformational changes in the penton base protein. Our findings provide the structural basis for adaptation to a fundamentally different tissue tropism of enteric HAdVs.

microbiology↗

bioRxiv2018

ATP-dependent force generation and membrane scission by ESCRT-III and Vps4

The ESCRTs catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated a minimal ESCRT module consisting of ESCRT-III subunits Snf7, Vps24, and Vps2, and the AAA+ ATPase Vps4 such that membrane nanotubes reflecting the correct topology of scission could be pulled from giant vesicles. Upon ATP release by photo-uncaging, this system was capable of generating forces within the nanotubes in a manner dependent upon Vps4 catalytic activity, Vps4 coupling to the ESCRT-III proteins, and membrane insertion by Snf7. At physiological concentrations, single scission events were observed that correlated with forces of ~6 pN, verifying predictions that ESCRTs are capable of exerting forces on membranes. Imaging of scission with subsecond resolution revealed Snf7 puncta at the sites of membrane cutting, directly verifying longstanding predictions for the ESCRT scission mechanism.\n\nOne Sentence SummaryESCRT-III and Vps4 were reconstituted from within the interior of nanotubes pulled from giant vesicles, revealing that this machinery couples ATP-dependent force production for membrane scission.

biophysics↗