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Attenuation hotspots in neurotropic human astroviruses [1]

['Hashim Ali', 'Department Of Pathology', 'University Of Cambridge', 'Addenbrookes Hospital', 'Cambridge', 'United Kingdom', 'Aleksei Lulla', 'Department Of Biochemistry', 'Alex S. Nicholson', 'Cambridge Institute For Medical Research']

Date: 2023-07

During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised, and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3′ end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics (RG) and replicon systems were developed. Recombinant truncated MLB viruses resulted in imbalanced RNA synthesis and strong attenuation in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals, and poultry.

Funding: This work was funded by a Sir Henry Dale Fellowship (220620/Z/20/Z) from the Wellcome Trust and the Royal Society, an Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge Joint Research Grant and MRC project grant (MR/T000376/1) to V.L. J.E.D and A.S.N. are supported by a Wellcome Trust Senior Research Fellowship (219447/Z/19/Z) awarded to J.E.D. R.L.O. is supported by the MRC DTP Studentship https://www.ukri.org/councils/mrc/ https://wellcome.org/ https://www.research-strategy.admin.cam.ac.uk/research-funding/internal-funding-opportunities/institutional-sponsorship-grants/wellcome-trust The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting Information files. Quantitative data can be found in the spreadsheet S1 Data . A separate tab is associated with each Figure and Supporting Information. Uncropped images are found in S1 Raw Images . GenBank accession numbers: ON398705, ON398706.

Copyright: © 2023 Ali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

It is therefore essential to develop the RG system for neurotropic astroviruses to understand the molecular determinants for neurotropism and neurovirulence. Here, we report the RG system for 2 nonclassical human neurotropic astroviruses that relies on a single cell line and can be used to rescue and propagate MLB1 and MLB2 human astroviruses. We also developed a set of detection tools as well as replicon systems for both MLB astroviruses. Using this system, we identified and characterized attenuation hotspots located at the 3′ end of the MLB genomes that impact neurovirulence of these viruses. In the future, this RG system will deepen the understanding of molecular virology of MLB-group astroviruses and allow the design of tools to address open questions on viral evolution, replication, packaging, and pathogenesis.

In 1997, Matsui’s group established the first RG system for the human astrovirus serotype 1 to rescue infectious viral particles [ 20 ]. This system has been successfully used and has shed light on multiple aspects of astrovirus replication and pathogenesis. HAstV1 RG system requires 2 cell lines to recover infectious particles: the transfection of BHK-21 cells with in vitro transcribed viral RNA and then propagation of the obtained supernatant in the permissive Caco-2 cells in the presence of trypsin. Several DNA-based RG systems were developed, including efficient chimeric HAstV1/8 RG system [ 21 , 22 ]; however, all of them relied on 2 cell lines and were limited to classical human astroviruses. So far, RG systems for 2 nonhuman astroviruses were developed: first for the avian astrovirus by using duck astrovirus (DAstV) genome of D51 strain [ 23 ] and second for porcine astrovirus (PAstV1-GX1) [ 24 ]. Although both nonhuman RG systems allow the recovery of infectious viral particles, these systems also rely on the 2 cell lines.

Growing evidence suggests that astroviruses are found globally, infecting a wide range of species, and have the potential for recombination, rapid evolution, and can adapt to different hosts [ 5 , 14 – 19 ]. Unfortunately, many astrovirus groups have remained overlooked for decades because of the absence of molecular tools, such as infectious clones and replicons. Therefore, developing a robust reverse genetics (RG) system for the nonclassical human astroviruses is essential to understand the basic biology, evolution, and host–virus interplay.

HAstVs are small, non-enveloped, icosahedral viruses with positive-sense single-stranded RNA genome containing 5′ untranslated region (UTR), 4 open reading frames (ORF1a, ORF1b, ORFX, and ORF2), and a 3′ UTR with poly A tail [ 6 , 7 ]. ORF1a encodes nonstructural polyprotein nsP1a, ORF1b is expressed via ribosomal frameshifting mechanism and encodes the RNA-dependent RNA polymerase (RdRp). The subgenomic (SG) RNA encodes 2 ORFs–ORF2 and ORFX, the latter encoding a viroporin [ 7 ]. The product of the ORF2 coding sequence is translated into the structural capsid polyprotein (CP) of about 72 to 90 kDa, depending on the virus strain [ 8 ], which then undergoes C-terminal cleavage by cellular caspases [ 9 ]. Despite the required function of caspases, the astrovirus release is described as an unclassified nonlytic process [ 10 ]. In some astroviruses, the structural polyprotein is cleaved by trypsin resulting in the formation of 3′ truncated (25 to 34 kDa) proteins. Maturation of the astrovirus capsid protein is a very dynamic process, transforming the virus from a noninfectious intracellular form (VP90) to a primed extracellular form (VP70) and finally generating an icosahedral infectious mature virion (VP34/27/25). However, some concerns remain to be addressed to fully understand the astroviruses capsid assembly and maturation. In particular, what differences in the capsid protein of the MLB genotypes make them different from the classical astroviruses and what determines their infectivity? It has been shown that trypsin treatment of classical astroviruses increases their infectivity [ 11 , 12 ], while not affecting the MLB genotypes [ 2 ], further confirmed by recently revealed differences in HAstV and MLB capsid structure [ 13 ]. The mechanism of CP cleavage and the functional role of cleaved CP in the MLB group of astroviruses is not yet understood. It is also unclear if MLB astroviruses exploit cellular proteases other than trypsin to process the capsid protein and how this impacts the infectivity of virus particles.

(A) Simplified phylogenetic tree for the Astrovirus genus. The tree is based on full nucleotide sequences available for indicated species. The pictogram of the intestine or neuron indicates the tropism associated with astrovirus strains. Some astrovirus genotypes labeled with the neuron icon are associated with neuropathology. Neurotropic MLB strains are shown in red. (B) The evolution experiment was performed for MLB1 and MLB2 astroviruses. (C) The coevolution experiment was performed for MLB1 and MLB2 astroviruses. (D) Nucleotide and amino acid sequences of MLB1 and MLB2 viruses containing deletions identified in evolved MLB virus stocks.

Human astroviruses (HAstVs) belong to the genus Mamastrovirus, family Astroviridae and are a common cause of gastroenteritis in children, the elderly, and immunocompromised adults [ 1 ]. Lately, the HAstV group of the Astroviridae family has expanded to include new groups of viruses unrelated to the 8 previously described classic HAstV serotypes ( Fig 1A ). These new human astrovirus groups are more closely related to certain animal astroviruses than to the classical HAstVs, suggesting zoonotic transmission [ 1 ]. One of these groups is named MLB, after the first novel human astrovirus described in 2008 in Melbourne (Australia) identified in feces of pediatric patients with gastroenteritis. Later, the MLB group of HAstVs was assigned to a neurovirulent group of astroviruses due to the association with severe cases of meningitis/encephalitis, febrile illness, and respiratory syndromes [ 2 – 4 ]. Interestingly, it was recently shown that astroviruses found in the fecal samples of macaque monkeys were genetically similar to human astrovirus MLB and caused chronic diarrhea [ 5 ].

Results

Evolution and cell culture adaptation of neurotropic MLB astroviruses The attenuation of viruses by serial cultivation in vitro or in abnormal hosts dates back to the 19th century [25]. Passaging pathogenic viruses in chicken embryos, mice and/or cell cultures led to the development of several vaccine candidates including polio, measles, yellow fever, rubella, and many other pathogens [26,27]. Therefore, we hypothesized that this strategy could be used to attenuate MLB astroviruses. Clinical MLB isolates were obtained from stool (MLB1) and cerebrospinal fluid (MLB2) of infected patients and passaged in susceptible cell lines, as previously described [2] (Fig 1B). Sequencing of a passaged clinical MLB1 isolate revealed a deletion of 30 nucleotides in the 3′ end of the genome spanning into the coding sequence of CP. A similar region was affected in the passaged MLB2 clinical isolate—a single out-of-frame deletion of 5 nucleotides in the 3′ part of the genome (Fig 1D). Another strategy for directed virus evolution is based on coinfection of closely related virus species [28]. The better replicating “partner” can either out-compete or complement the replication of another virus. Complementation would result in faster evolution of less “fit” viruses. To test this hypothesis, we coinfected Huh7.5.1 cells at a multiplicity of infection (MOI) 0.1 with MLB1 and MLB2 viruses (Fig 1C). This strategy can facilitate complementation after the first cycle of virus replication while avoiding the accumulation of defective interfering genomes common for the high MOI [29]. This resulted in simultaneous replication and propagation of both strains on passaging without detected out-competition or recombination for 10 consecutive passages. Interestingly, no changes were observed in MLB2 genomes; however, a mixture of several in-frame and out-of-frame deletions was detected in the 3′ part of the MLB1 genome further confirming the instability of the 3′ region in this virus (Fig 1D). Elucidating the functional significance of the identified deletions requires MLB astrovirus detection tools and would be dramatically accelerated by the establishment of a robust RG system. We, therefore, aimed to create these essential tools.

Cell culture models and detection tools for neurotropic MLB1 and MLB2 astroviruses First, we developed a set of essential tools for specific immune detection of virus infection. The folded region of MLB1 capsid protein corresponding to amino acids 61–396 of ORF2-encoded polyprotein possessing a C-terminal 8×His-tag (Fig 2A) was used for bacterial expression and affinity purification, resulting in homogeneous CP NTD protein (Fig 2B). The purified recombinant protein was used for the production of highly sensitive antibodies allowing the detection of ≤1 ng of the purified CP NTD of MLB1 (Fig 2C). Due to 95% identity between corresponding domains of MLB1 and MLB2 CPs, polyclonal antibodies were expected to cross-detect capsid proteins derived from both strains. Indeed, it specifically recognized capsid proteins from MLB1- and MLB2-infected cells (Fig 2C). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Purification of CP NTD and immunodetection of MLB1- and MLB2-infected Huh7.5.1 cells and iPSC-derived neurons. (A) Schematic representation of MLB1 genome and location of CP NTD . Lower panel represents the sequence of recombinant CP NTD . ORF, open reading frame; CP, capsid protein; NTD, N-terminal domain. (B) Coomassie-stained SDS-PAGE profile of the purified CP NTD from E. Coli. (C) Huh7.5.1 cells were infected with MLB1 and MLB2 viruses at MOI 0.1 and incubated for 48 h. CP was detected using the antibody generated against CP NTD . Purified CP NTD was used for detection limit assessment (1–9 ng). (D) Experimental setup to determine the CPE and titration for MLB1 and MLB2 infection. (E) Huh7.5.1 cells were infected at an MOI 1 and incubated for indicated periods, then stained and imaged. Hoechst-stained nuclei were counted from 12 images (approximately 200 cells per image) and normalized to mock-infected samples. Data are mean ± SD. ***p < 0.001, ****p < 0.0001 using two-way ANOVA test against mock. (F) Huh7.5.1 cells were seeded on 96-well plate and infected with 10-fold serial dilutions of MLB1 and MLB2 astroviruses, fixed at 20 hpi, permeabilized, stained with anti-CP antibody, and imaged by LI-COR. (G) Huh7.5.1 cells were infected with MLB1 and MLB2 viruses and incubated for 24 h. Representative confocal images of fixed and permeabilized cells visualized for CP (green) and stained for nuclei (Hoechst, blue) are shown. Scale bars are 10 μm. (H) Huh7.5.1 cells were infected with MLB1 and MLB2 virus stocks at MOI 0.1 and incubated for 72–120 h. Virus titers were determined from 6 independent experiments. Data are mean ± SD. (I) Partially differentiated i3Neurons were seeded on IBIDI plates, differentiated into mature glutamatergic neurons, infected with MLB1 and MLB2 viruses, and incubated for 48 (MLB2) or 96 (MLB1) h. Representative confocal images of fixed and permeabilized cells visualized for MLB CP (green), neuronal marker MAP2 (magenta) and stained for nuclei (Hoechst, blue) are shown. Scale bars are 50 μm. All uncropped images can be found in the Supporting information file as S1 Raw Images. All individual quantitative observations that underlie the data can be found in S1 Data file. CPE, cytopathic effect; hpi, hours post infection; MOI, multiplicity of infection. https://doi.org/10.1371/journal.pbio.3001815.g002 When passaging clinical isolates, we noticed that the Huh7.5.1 cell line supports MLB replication and results in a moderate cytopathic effect (CPE). MLB clade viruses were previously reported to replicate in Huh7 and Huh7.5 cell lines with the ability to establish a persistent infection on passaging [2]. Presumably, in contrast to the immune-competent Huh7 cells, the more susceptible Huh7.5.1 cell line [7] can allow for enhanced replication and development of CPE. This cell line was also reported to support active replication of the classical human astrovirus 1 (HAstV1) [30]. The CPE was apparent for MLB2 at 24 to 48 h post infection (hpi), whereas slower replicating MLB1 showed CPE at 48 to 72 hpi, reaching >60% cell death at 3 days post infection for MLB2 and at 4 days post infection for MLB1 (Fig 2D and 2E). The differences in CPE were also confirmed in the independent automated cytotoxicity screening using live-cell imaging (S1 Fig). The permissiveness of the Huh7.5.1 cell line allowed the development of a virus titration system. Cells cultured on 96-well plates were infected with 10-fold dilutions of MLB1 and MLB2 stocks, fixed and stained with CP NTD -antibody using in-cell near-infrared fluorescence-based detection (Fig 2D and 2F). The cytoplasmic distribution of CP was confirmed by confocal microscopy further demonstrating that both MLB1 and MLB2 can be detected 24 hpi (Fig 2G). Since no released virus could be detected after 20 hpi, this timepoint was utilized for single-round infection experiments like titration of the virus stocks. Efficient virus release was detected at 48 to 72 hpi for MLB2 and at 96 to 120 hpi for MLB1, reaching 1–3 × 106 infectious units (IU) per ml (Fig 2H). Finally, to confirm the neurotropic properties of MLB astroviruses, we developed a physiologically relevant system to infect and monitor MLB infection in neurons. The neurotropism of MLB astroviruses was previously described and indicates their ability to infect cells of neuronal origin [2,4]. The recently developed methodology to efficiently differentiate human-induced pluripotent stem cells (iPSCs) into isogenic cortical glutamatergic neurons (i3Neurons) [31] provided a suitable platform to experimentally assess neurotropic properties of MLB1 and MLB2 viruses. Both viruses resulted in efficient infection at 48 hpi (MLB2) and 96 hpi (MLB1) further confirming the ability of these viruses to infect postmitotic neuronal cells (Fig 2I).

Assessing genome stability of MLB1 and MLB2 viruses To evaluate the MLB virus genome stabilities, we performed serial passaging by using RG-derived MLB1 and MLB2 recombinant viruses. The passaging was performed in biological duplicate, starting from in vitro RNA transcripts. No changes were detected in the passaged recombinant MLB2 virus, suggesting that its genome is stable in Huh7.5.1 cells. The slower replicating recombinant MLB1 was less stable and accumulated mutations in the 3′ part of the genome. An out-of-frame single-nucleotide insertion was detected at passage 3, that coexisted with wild-type (wt) MLB1 resulting in continuous coinfection for 7 consecutive passages (Table 1). A distinct cluster of mutations at the C-terminal end of CP was identified in the second experiment (Table 1) suggesting instability of RG MLB1 genomes during longer virus passaging. PPT PowerPoint slide

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TIFF original image Download: Table 1. Genome changes in evolved recombinant MLB1 and MLB2 astroviruses. https://doi.org/10.1371/journal.pbio.3001815.t001 To put these mutations and previously identified deletions (Fig 1D) in the context of naturally occurring changes in MLB genomes, the analysis of all available GenBank MLB astrovirus sequences was performed. As expected, the 3′ region of the MLB2 genome was very conserved: Few amino acid variations and no deletions were found in publicly available MLB2 genome sequences (Fig 4A). In contrast, the 3′ region of MLB1 was more diverse, containing multiple changes throughout the analyzed region as well as 1 amino acid deletion upstream of the experimentally observed deletion region (Fig 4B), consistent with the mutation- and deletion-prone nature of the 3′ region of MLB1 genome. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Analysis of C-terminal part of MLB1 and MLB2 astrovirus CP. (A) Analysis of publicly available sequences of C-terminal part of MLB2 CP. (B) Analysis of publicly available sequences of C-terminal part of MLB1 CP. The deletion region is indicated on top of MLB1 and MLB2 alignments (A, B). (C) Analysis of the C-terminal part of MLB1 and MLB2 CP for putative caspase cleavage sites using Procleave software [33]. The sequences in bold indicate caspase 1, 3, and 6 putative cleavage sites with a probability score of >0.7. The regions of MLB1 (top) and MLB2 (bottom) deletions are shown. (D) Huh7.5.1 cells were infected with indicated recombinant viruses of MLB1 (left) and MLB2 (right) at an MOI 0.5 in triplicates, the virus was collected at indicated times post infection, and titer was measured for both extracellular and intracellular fractions. Data are mean ± SD. (E) Analysis of CP expression in Huh7.5.1 cells infected with second passage of MLB1 and MLB2 wt and mutant viruses (MOI 0.1). Cell lysates were harvested at 48 hpi and analyzed by western blotting with anti-CP and anti-tubulin antibodies. GNN is RdRp knock-out recombinant virus (GDD to GNN). (F) The effect of pan-caspase inhibitor z-VAD-fmk on CP processing during infection with classical human astrovirus 4 (HAstV4), MLB1 and MLB2 astroviruses. Caco2 cells were infected with HAstV4 and Huh7.5.1 cells were infected with MLB1 and MLB2 astroviruses at MOI 1 in the presence or absence of z-VAD-fmk. Cell lysates were harvested at indicated hpi and analyzed by western blotting with 8E7 antibody against HAstV CP (for HAstV4), anti-CP (for MLB), and anti-tubulin antibodies. The average inhibition of CP cleavage was quantified from 3 independent experiments. (G) The schematic representation of the virus competition experiment where Huh7.5.1 cells were coinfected with wt and mutant viruses and passaged. Virus RNA was isolated and used for RT-PCR to detect virus-specific fragments. (H) The RT-PCR fragments from the competition experiment were analyzed by agarose electrophoresis. The fragments corresponding to the expected size of each PCR product are shown on the right. All uncropped images can be found in the Supporting information file as S1 Raw Images. All individual quantitative observations that underlie the data can be found in S1 Data file. CP, capsid polyprotein; hpi, hours post infection; MOI, multiplicity of infection; wt, wild-type. https://doi.org/10.1371/journal.pbio.3001815.g004

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