ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication.

The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.

COVID-19 in non-hospitalised adults caused by either SARS-CoV-2 sub-variants Omicron BA.1, BA.2, BA.4/5 or Delta associates with similar illness duration, symptom severity and viral kinetics, irrespective of vaccination history.

BACKGROUND: SARS-CoV-2 variant Omicron rapidly evolved over 2022, causing three waves of infection due to sub-variants BA.1, BA.2 and BA.4/5. We sought to characterise symptoms and viral loads over the course of COVID-19 infection with these sub-variants in otherwise-healthy, vaccinated, non-hospitalised adults, and compared data to infections with the preceding Delta variant of concern (VOC). METHODS: In a prospective, observational cohort study, healthy vaccinated UK adults who reported a positive polymerase chain reaction (PCR) or lateral flow test, self-swabbed on alternate weekdays until day 10. We compared participant-reported symptoms and viral load trajectories between infections caused by VOCs Delta and Omicron (sub-variants BA.1, BA.2 or BA.4/5), and tested for relationships between vaccine dose, symptoms and PCR cycle threshold (Ct) as a proxy for viral load using Chi-squared (χ2) and Wilcoxon tests. RESULTS: 563 infection episodes were reported among 491 participants. Across infection episodes, there was little variation in symptom burden (4 [IQR 3-5] symptoms) and duration (8 [IQR 6-11] days). Whilst symptom profiles differed among infections caused by Delta compared to Omicron sub-variants, symptom profiles were similar between Omicron sub-variants. Anosmia was reported more frequently in Delta infections after 2 doses compared with Omicron sub-variant infections after 3 doses, for example: 42% (25/60) of participants with Delta infection compared to 9% (6/67) with Omicron BA.4/5 (χ2 P < 0.001; OR 7.3 [95% CI 2.7-19.4]). Fever was less common with Delta (20/60 participants; 33%) than Omicron BA.4/5 (39/67; 58%; χ2 P = 0.008; OR 0.4 [CI 0.2-0.7]). Amongst infections with an Omicron sub-variants, symptoms of coryza, fatigue, cough and myalgia predominated. Viral load trajectories and peaks did not differ between Delta, and Omicron, irrespective of symptom severity (including asymptomatic participants), VOC or vaccination status. PCR Ct values were negatively associated with time since vaccination in participants infected with BA.1 (ß = -0.05 (CI -0.10-0.01); P = 0.031); however, this trend was not observed in BA.2 or BA.4/5 infections. CONCLUSION: Our study emphasises both the changing symptom profile of COVID-19 infections in the Omicron era, and ongoing transmission risk of Omicron sub-variants in vaccinated adults. TRIAL REGISTRATION: NCT04750356.

Combined analyses of within-host SARS-CoV-2 viral kinetics and information on past exposures to the virus in a human cohort identifies intrinsic differences of Omicron and Delta variants.

The emergence of successive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) during 2020 to 2022, each exhibiting increased epidemic growth relative to earlier circulating variants, has created a need to understand the drivers of such growth. However, both pathogen biology and changing host characteristics-such as varying levels of immunity-can combine to influence replication and transmission of SARS-CoV-2 within and between hosts. Disentangling the role of variant and host in individual-level viral shedding of VOCs is essential to inform Coronavirus Disease 2019 (COVID-19) planning and response and interpret past epidemic trends. Using data from a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening, we developed a Bayesian hierarchical model to reconstruct individual-level viral kinetics and estimate how different factors shaped viral dynamics, measured by PCR cycle threshold (Ct) values over time. Jointly accounting for both interindividual variation in Ct values and complex host characteristics-such as vaccination status, exposure history, and age-we found that age and number of prior exposures had a strong influence on peak viral replication. Older individuals and those who had at least 5 prior antigen exposures to vaccination and/or infection typically had much lower levels of shedding. Moreover, we found evidence of a correlation between the speed of early shedding and duration of incubation period when comparing different VOCs and age groups. Our findings illustrate the value of linking information on participant characteristics, symptom profile and infecting variant with prospective PCR sampling, and the importance of accounting for increasingly complex population exposure landscapes when analysing the viral kinetics of VOCs. Trial Registration: The Legacy study is a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening for SARS-CoV-2 at University College London Hospitals or at the Francis Crick Institute (NCT04750356) (22,23). The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469). The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469) and is sponsored by University College London Hospitals. Written consent was given by all participants.

Within-host SARS-CoV-2 viral kinetics informed by complex life course exposures reveals different intrinsic properties of Omicron and Delta variants.

The emergence of successive SARS-CoV-2 variants of concern (VOC) during 2020-22, each exhibiting increased epidemic growth relative to earlier circulating variants, has created a need to understand the drivers of such growth. However, both pathogen biology and changing host characteristics - such as varying levels of immunity - can combine to influence replication and transmission of SARS-CoV-2 within and between hosts. Disentangling the role of variant and host in individual-level viral shedding of VOCs is essential to inform COVID-19 planning and response, and interpret past epidemic trends. Using data from a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening, we developed a Bayesian hierarchical model to reconstruct individual-level viral kinetics and estimate how different factors shaped viral dynamics, measured by PCR cycle threshold (Ct) values over time. Jointly accounting for both inter-individual variation in Ct values and complex host characteristics - such as vaccination status, exposure history and age - we found that age and number of prior exposures had a strong influence on peak viral replication. Older individuals and those who had at least five prior antigen exposures to vaccination and/or infection typically had much lower levels of shedding. Moreover, we found evidence of a correlation between the speed of early shedding and duration of incubation period when comparing different VOCs and age groups. Our findings illustrate the value of linking information on participant characteristics, symptom profile and infecting variant with prospective PCR sampling, and the importance of accounting for increasingly complex population exposure landscapes when analysing the viral kinetics of VOCs.

The flavivirus polymerase NS5 regulates translation of viral genomic RNA.

Flaviviruses, including dengue virus and Zika virus, contain a single-stranded positive sense RNA genome that encodes viral proteins essential for replication and also serves as the template for new genome synthesis. As these processes move in opposite directions along the genome, translation must be inhibited at a defined point following infection to clear the template of ribosomes to allow efficient replication. Here, we demonstrate in vitro and in cell-based assays that the viral RNA polymerase, NS5, inhibits translation of the viral genome. By reconstituting translation in vitro using highly purified components, we show that this translation block occurs at the initiation stage and that translation inhibition depends on NS5-RNA interaction, primarily through association with the 5' replication promoter region. This work supports a model whereby expression of a viral protein signals successful translation of the infecting genome, prompting a switch to a ribosome depleted replication-competent form.

Mouse Ifit1b is a cap1-RNA-binding protein that inhibits mouse coronavirus translation and is regulated by complexing with Ifit1c.

Knockout mouse models have been extensively used to study the antiviral activity of IFIT (interferon-induced protein with tetratricopeptide repeats). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of "self" in higher eukaryotes, whereas unmodified cap0-RNA is recognized as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess "nonself" cap0-mRNAs. However, in mice and other rodents, the IFIT1 orthologue has been lost, and the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b, and Ifit1c. Although murine Ifit1 is similar to human IFIT1 in its cap0-RNA-binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, whereas binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict WT mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families to facilitate better design and interpretation of mouse models of human infection and sheds light on the evolutionary plasticity of the IFIT family.

Circularization of flavivirus genomic RNA inhibits de novo translation initiation.

Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA-RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome.

Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells.

Background: Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Methods: Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro. Results: Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Conclusions: Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.

Better together: the role of IFIT protein-protein interactions in the antiviral response.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of antiviral proteins conserved throughout all vertebrates. IFIT1 binds tightly to non-self RNA, particularly capped transcripts lacking methylation on the first cap-proximal nucleotide, and inhibits their translation by out-competing the cellular translation initiation apparatus. This exerts immense selection pressure on cytoplasmic RNA viruses to maintain mechanisms that protect their messenger RNA from IFIT1 recognition. However, it is becoming increasingly clear that protein-protein interactions are necessary for optimal IFIT function. Recently, IFIT1, IFIT2 and IFIT3 have been shown to form a functional complex in which IFIT3 serves as a central scaffold to regulate and/or enhance the antiviral functions of the other two components. Moreover, IFITs interact with other cellular proteins to expand their contribution to regulation of the host antiviral response by modulating innate immune signalling and apoptosis. Here, we summarize recent advances in our understanding of the IFIT complex and review how this impacts on the greater role of IFIT proteins in the innate antiviral response.

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.