The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

AbstractProgrammed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

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Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

10.1101/2021.05.31.445667 ◽

2021 ◽

Author(s):

Matthias Michael Zimmer ◽

Anuja Nitin Kibe ◽

Ulfert Rand ◽

Lukas Pekarek ◽

Luca Cicin-Sain ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Specific Inhibitor ◽

Antiviral Protein ◽

Ribosomal Frameshifting ◽

Reading Frame ◽

Rna Molecules ◽

Cellular Factors ◽

Infected Cells

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

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Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

10.21203/rs.3.rs-563757/v1 ◽

2021 ◽

Author(s):

Matthias Zimmer ◽

Anuja Kibe ◽

Ulfert Rand ◽

Lukas Pekarek ◽

Luka Cicin-Sain ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Specific Inhibitor ◽

Antiviral Protein ◽

Ribosomal Frameshifting ◽

Reading Frame ◽

Rna Molecules ◽

Cellular Factors ◽

Infected Cells

Abstract Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

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The energy landscape of −1 ribosomal frameshifting

Science Advances ◽

10.1126/sciadv.aax6969 ◽

2020 ◽

Vol 6 (1) ◽

pp. eaax6969 ◽

Cited By ~ 13

Author(s):

Junhong Choi ◽

Sinéad O’Loughlin ◽

John F. Atkins ◽

Joseph D. Puglisi

Keyword(s):

Single Molecule ◽

Messenger Rna ◽

Information Transfer ◽

Mechanistic Model ◽

High Rate ◽

Coding Region ◽

Ribosomal Frameshifting ◽

Reading Frame

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3′ messenger RNA (mRNA) structures, but how these mRNA structures enhance −1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal −1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

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New lane in the information highway: alternative reading frame peptides elicit T cells with potent antiretrovirus activity

Journal of Experimental Medicine ◽

10.1084/jem.20071986 ◽

2007 ◽

Vol 204 (11) ◽

pp. 2501-2504 ◽

Cited By ~ 9

Author(s):

Jonathan W. Yewdell ◽

Heather D. Hickman

Keyword(s):

T Cells ◽

Open Reading Frames ◽

Viral Escape ◽

Antigenic Peptides ◽

Reading Frame ◽

Alternative Reading Frame ◽

Cell Responses ◽

Infected Cells ◽

Retroviral Replication ◽

Alternative Reading

CD8+ T cells rapidly recognize virus-infected cells due to the generation of antigenic peptides from defective ribosomal products (DRiPs) that are encoded by standard open reading frames (ORFs). New data now show that alternative reading frame (ARF) DRiPs can also induce robust CD8+ T cell responses. ARF-specific T cells control retroviral replication and select for viral escape in monkeys, providing the most compelling evidence to date for the biological relevance of ARF immunosurveillance.

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Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro

Journal of Virology ◽

10.1128/jvi.72.3.2265-2271.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 2265-2271 ◽

Cited By ~ 42

Author(s):

Xiao Tao Lu ◽

Amy C. Sims ◽

Mark R. Denison

Keyword(s):

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Open Reading Frame ◽

Cleavage Sites ◽

Reading Frame ◽

Amino Terminal ◽

Infected Cells ◽

Carboxy Terminal ◽

A Site

ABSTRACT The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

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Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

10.1101/2020.09.11.294280 ◽

2020 ◽

Cited By ~ 1

Author(s):

Werner J. D. Ouwendijk ◽

Daniel P. Depledge ◽

Labchan Rajbhandari ◽

Tihana Lenac Rovis ◽

Stipan Jonjic ◽

...

Keyword(s):

Gene Expression ◽

Varicella Zoster Virus ◽

Viral Gene Expression ◽

Fusion Transcript ◽

Viral Gene ◽

Reading Frame ◽

Varicella Zoster ◽

Rapid Amplification

SummaryVaricella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5’ rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

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Human Cytomegalovirus Gene Expression Is Silenced by Daxx-Mediated Intrinsic Immune Defense in Model Latent Infections Established In Vitro

Journal of Virology ◽

10.1128/jvi.00827-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9109-9120 ◽

Cited By ~ 109

Author(s):

Ryan T. Saffert ◽

Robert F. Kalejta

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Chromatin Structure ◽

Lytic Replication ◽

Immune Defense ◽

Latent Infections ◽

Abortive Infection ◽

Latently Infected ◽

Infected Cells

ABSTRACT In addition to productive lytic infections, herpesviruses such as human cytomegalovirus (HCMV) establish a reservoir of latently infected cells that permit lifelong colonization of the host. When latency is established, the viral immediate-early (IE) genes that initiate the lytic replication cycle are not expressed. HCMV IE gene expression at the start of a lytic infection is facilitated by the viral pp71 protein, which is delivered to cells by infectious viral particles. pp71 neutralizes the Daxx-mediated cellular intrinsic immune defense that silences IE gene expression by generating a repressive chromatin structure on the viral major IE promoter (MIEP). In naturally latently infected cells and in cells latently infected in vitro, the MIEP also adopts a similar silenced chromatin structure. Here we analyze the role of Daxx in quiescent HCMV infections in vitro that mimic some, but not all, of the characteristics of natural latency. We show that in these “latent-like” infections, the Daxx-mediated defense that represses viral gene expression is not disabled because pp71 and Daxx localize to different cellular compartments. We demonstrate that Daxx is required to establish quiescent HCMV infections in vitro because in cells that would normally foster the establishment of these latent-like infections, the loss of Daxx causes the lytic replication cycle to be initiated. Importantly, the lytic cycle is inefficiently completed, which results in an abortive infection. Our work demonstrates that, in certain cell types, HCMV must silence its own gene expression to establish quiescence and prevent abortive infection and that the virus usurps a Daxx-mediated cellular intrinsic immune defense mechanism to do so. This identifies Daxx as one of the likely multiple viral and cellular determinants in the pathway of HCMV quiescence in vitro, and perhaps in natural latent infections as well.

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Alternate RNA splicing of murine nfkb1 generates a nuclear isoform of the p50 precursor NF-kappa B1 that can function as a transactivator of NF-kappa B-regulated transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8460 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8460-8470 ◽

Cited By ~ 15

Author(s):

R J Grumont ◽

J Fecondo ◽

S Gerondakis

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Rna Splicing ◽

Coding Region ◽

Nf Kappa B ◽

Reading Frame ◽

C Terminus ◽

A Cell ◽

Regulated Gene Expression

The NF-kappa B1 subunit of the transcription factor NF-kappa B is derived by proteolytic cleavage from the N terminus of a 105-kDa precursor protein. The C terminus of p105NF-kappa B1, like those of I kappa B proteins, contains ankyrin-related repeats that inhibit DNA binding and nuclear localization of the precursor and confer I kappa B-like properties upon p105NF-kappa B1. Here we report the characterization of two novel NF-kappa B1 precursor isoforms, p84NF-kappa B1 and p98NF-kappa B1, that arise by alternate splicing within the C-terminal coding region of murine nfkb1. p98NF-kappa B1, which lacks the 111 C-terminal amino acids (aa) of p105NF-kappa B1, has a novel 35-aa C terminus encoded by an alternate reading frame of the gene. p84NF-kappa B1 lacks the C-terminal 190 aa of p105NF-kappa B1, including part of ankyrin repeat 7. RNA and protein analyses indicated that the expression of p84NF-kappa B1 and p98NF-kappa B1 is restricted to certain tissues and that the phorbol myristate acetate-mediated induction of p84NF-kappa B1 and p105NF-kappa B1 differs in a cell-type-specific manner. Both p84NF-kappa B1 and p98NF-kappa B1 are found in the nuclei of transfected cells. Transient transfection analysis revealed that p98NF-kappa B1, but not p105NF-kappa B1 or p84NF-kappa B1, acts as a transactivator of NF-kappa B-regulated gene expression and that this is dependent on sequences in the Rel homology domain required for DNA binding and on the novel 35 C-terminal aa of this isoform. In contrast to previous findings, which indicated that p105NF-kappa B1 does not bind DNA, all of the NF-kappa B1 precursors were found to specifically bind with low affinity to a highly restricted set of NF-kappa B sites in vitro, thereby raising the possibility that certain of the NF-kappa B1 precursor isoforms may directly modulate gene expression.

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Murid Herpesvirus 4 Strain 68 M2 Protein Is a B-Cell-Associated Antigen Important for Latency but Not Lymphocytosis

Journal of Virology ◽

10.1128/jvi.77.17.9700-9709.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9700-9709 ◽

Cited By ~ 38

Author(s):

Alastair I. Macrae ◽

Edward J. Usherwood ◽

S. Mazher Husain ◽

Emilio Flaño ◽

In-Jeong Kim ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Cell Receptor ◽

Cell Number ◽

Reading Frame ◽

Latently Infected ◽

Infected Cells ◽

Herpesvirus 4 ◽

Productive Replication

ABSTRACT This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vβ4 T-cell-receptor-positive CD8+ T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.

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