The role of -2/-1 ribosome frameshifting mechanism in PRRS virus replication

Yan, Xingyu

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https://hdl.handle.net/2142/121359 Copy

Description

Title

The role of -2/-1 ribosome frameshifting mechanism in PRRS virus replication

Author(s)

Yan, Xingyu

Issue Date

2023-07-13

Director of Research (if dissertation) or Advisor (if thesis)

Fang, Ying

Doctoral Committee Chair(s)

Fang, Ying

Committee Member(s)

• Rowland, Raymond
• Smith, Rebecca Lee
• Dilger, Anna

Department of Study

Pathobiology

Discipline

VMS - Pathobiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

PRRSV, ribosome frameshifting, viral pathogenesis

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically important swine disease worldwide. Understanding the replication mechanisms of PRRSV is of great significance to design novel vaccines or antiviral strategies. As a member of the Nidovirales, PRRSV utilizes a canonical -1 programmed ribosomal frameshifting (PRF) to express open reading frame 1b (ORF1b), while a unique -2/-1 PRF was later identified in the nonstructural protein 2 (nsp2)-coding region, which results in the generation of 2 novel viral proteins nsp2TF and nsp2N. The knockout of these frameshifting products resulted in significantly impaired viral growth kinetics, indicating their crucial role during PRRSV viral replication. The functions of nsp2TF were further explored in chapter 2, and the exact function of nsp2N is currently unknown. In chapter 2, we characterized the unique function of the PRRSV -2 PRF product, nsp2TF, as a deubiquitinase to antagonize the poly-ubiquitination of PRRSV GP5 and M protein and thus promoting the virus assembly. In PRRSV-infected cells, GP5 and M protein were found to be colocalized with nsp2TF instead of nsp2. Co-immunoprecipitation results further confirmed the direct interaction between nsp2TF and GP5/M. GP5 and M protein were poly-ubiquitinated in infected cells and over-expression system, while the co-expression of nsp2TF could reverse such ubiquitination due to its papain-like protease domain (PLP2). The nsp2TF-knockout mutant (vKTF) showed significantly impaired growth kinetics and the GP5/M expression level decreased gradually compared with parental virus. All these data suggested that nsp2TF functions as a deubiquitinase to stabilize GP5/M proteins and promote viral replication. In chapter 3, the key residues of PRRSV nsp1beta that could significantly affect -2/-1 PRF were identified. Unlike the canonical -1 ribosomal frameshifting in Nidoviruses, arteriviruses utilize viral RNA-viral protein (nsp1beta)-host protein (PCBPs) complex to initiate -2/-1 PRF. Our results showed that the residues Y115 and G120 (the relative amino acid positions of PRRSV-2 nsp1beta) which are highly conserved in arteriviruses almost completely abolish the -2/-1 PRF in PRRSV-2. By introducing the single mutation of each residue (Y115A or G120A) into the infectious clone, both mutants showed significantly impaired growth kinetics compared with the parental virus. Further characterizations demonstrated that these two residues play a key role for nsp1beta to interact with host factors PCBP1 and PCBP2. Co-immunoprecipitation results showed that the amounts of PCBP1 and PCBP2 pulled down by nsp1beta with mutation Y115A or G120A were significantly lower than those pulled down by the wild type (WT) nsp1beta. Our data suggested that Y115 and G120 of nsp1beta could significantly affect PRRSV -2/-1 PRF and viral replication as they are crucial to maintain sufficient nsp1beta-PCBPs interaction. In chapter 4, a recently emerging PRRSV variant which caused severe clinical disease was isolated and named as UIL21-0712. The sequence analysis revealed that this isolate has a 1-4-4 restriction fragment length polymorphism (RFLP) cutting pattern and belongs to PRRSV lineage 1c. The PRRSV RFLP 1-4-4 L1c variant first emerged in the middle U.S in early 2020 and the first outbreak occurred in the end of the year. Currently, it is still the predominant variant circulating in the field. Our previous study already identified that this emerging variant possesses distinct nucleotide substitutions in the -2/-1 PRF region, which results in the extension of the -1 PRF product, nsp2N. In this study, we further characterized this variant by comparing its nucleotide (nt) and amino acid (aa) sequence identities of each individual antigens, as well as all the identified B cell or T cell epitopes, with four commercially available vaccine strains in order to select the ideal vaccine against the outbreaking variant. Our data suggested that there is no single optimal vaccine strain against RFLP 1-4-4 variant based on the sequence identity. In summary, our study revealed the roles of -2/-1 PRF mechanism in PRRS viral replication and the intriguing association of nsp2TF with GP5/M may provide novel insights for subunit vaccine development against PRRSV in the future.

Graduation Semester

2023-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Xingyu Yan

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