Recombinant Porcine reproductive and respiratory syndrome virus Envelope small membrane protein (GP2b)

What is the PRRSV envelope protein GP2b/2b/E?

The PRRSV 2b (also designated as E) protein is a small, non-glycosylated, hydrophobic protein of approximately 73 amino acids, encoded by an internal open reading frame (ORF) within the bicistronic mRNA2 of PRRSV. It functions as a minor structural component of the PRRSV virion. The designation "2b" is used by some researchers to avoid confusion with the use of "E" by others to identify the major envelope glycoprotein of PRRSV, GP5 . The protein is similar to the E protein of Equine Arteritis Virus (EAV), another member of the Arteriviridae family .

What is the molecular weight and abundance of the 2b/E protein in virions?

The 2b/E protein has a molecular weight of approximately 10 kDa as determined by SDS-PAGE analysis of radiolabeled virions . It is considered a minor structural component of the virus particle, with significantly lower abundance compared to major structural proteins. Experimental evidence shows that when comparing the 10 kDa band (2b) with the 15 kDa N protein band in purified virions, the 2b protein appears much fainter despite having a similar number of methionine and cysteine residues, suggesting fewer 2b molecules are incorporated into each virion .

What is the essential function of 2b/E protein in the PRRSV life cycle?

The 2b/E protein is essential for PRRSV infection but dispensable for virion assembly. When the E gene expression is blocked in a full-length infectious clone by mutating the ATG translational initiation codon to GTG, the resulting E protein-negative virus particles (P129-ΔE) are non-infectious despite being structurally similar to wild-type virions and containing viral genomic RNA . Experimental evidence indicates that while these E-deficient particles can enter cells, the subsequent steps of replication are interrupted. The protein appears to function as an ion-channel embedded in the viral envelope that facilitates uncoating of the virus and release of the genome in the cytoplasm .

How do researchers experimentally demonstrate that 2b/E is a structural component of the PRRSV virion?

Researchers have employed multiple complementary approaches to definitively establish that the 2b/E protein is an integral component of the PRRSV virion:

• Two-step purification with autoradiography:

  • First, [³⁵S]-methionine/cysteine-labeled virus is purified by sucrose-gradient centrifugation

  • Second, virions are captured using monoclonal antibody 2C12 (which recognizes the matrix protein exposed on the virus surface)

  • SDS-PAGE analysis reveals the presence of a 10 kDa protein (corresponding to 2b) along with the three major structural proteins N, M, and GP5

• Immunoprecipitation with anti-2b monoclonal antibody:

  • Development of monoclonal antibody 17C3 specific to the C-terminal peptide (amino acids 60-73) of the 2b protein

  • Using this antibody to immunoprecipitate 2b from sucrose-gradient purified virus preparations

  • Confirmation of specificity through competitive ELISA assays

These methodological approaches provide robust evidence that 2b is a minor but integral component of the PRRSV virion structure.

What evidence supports the ion-channel function of the 2b/E protein?

Several experimental observations support the hypothesis that the PRRSV 2b/E protein functions as a viroporin (viral ion channel):

• Growth inhibition in E. coli: Expression of the E protein in Escherichia coli results in cell growth arrest .

• Increased membrane permeability: E protein expression increases cell membrane permeability, consistent with pore-forming activity .

• Oligomerization capacity: Cross-linking experiments in PRRSV-infected cells or cells transfected with the E gene demonstrate that the E protein forms homo-oligomers, a characteristic feature of ion channel proteins .

• Pharmacological inhibition profile: PRRSV replication is effectively inhibited by lysomotropic basic compounds and known ion-channel blocking agents during the uncoating process, suggesting a functional relationship between ion channel activity and viral uncoating .

• Phenotype of E-deficient virions: E protein-negative virions can assemble and enter cells but fail to complete subsequent replication steps, indicating the protein's role in a post-entry step such as genome release .

How does the oligomerization of 2b/E protein contribute to its function?

The 2b/E protein forms homo-oligomers, as demonstrated through cross-linking experiments in both PRRSV-infected cells and cells transfected with the E gene . This oligomerization is likely critical for the protein's putative ion channel activity. Many viral ion channel proteins function by assembling into multimeric complexes that create a pore through the lipid bilayer. The oligomerization of 2b/E monomers likely creates a properly sized and shaped channel that allows specific ions to pass through the viral membrane during the uncoating process, facilitating genome release into the cytoplasm. The specific stoichiometry of these oligomers (whether they form tetramers, pentamers, or other structures) has not been definitively established in the provided research but would be a valuable avenue for future structural studies.

What is the significance of recombination in PRRSV evolution?

Recombination is a major contributor to PRRSV genetic diversity and evolution. Analysis of 949 PRRSV-2 genomic sequences collected from 1991 to 2021 revealed extensive interlineage recombination . Recombination serves as an important driver of genetic shifts that contribute to:

• The emergence of new PRRSV variants with altered phenotypes

• Changes in virulence and pathogenesis

• Potential vaccine escape

• Adaptation to new host populations

The temporal and geographical distribution of recombinant PRRSV strains shows increasing prevalence over time, with distinct patterns in different countries. For example, in China, recombinant PRRSV-2 was first detected in 2000, with significant increases in recombinant strains observed after 2014, while in the United States, recombinant strains markedly increased after 2012 .

What are the recombination hotspots in the PRRSV genome and how do they relate to the 2b/E protein?

Analysis of PRRSV-2 recombination patterns has identified specific genomic regions that are more prone to recombination events. One significant recombination hotspot region corresponds to nucleotides 682-801 in the NSP9 gene, which encodes part of the viral RNA-dependent RNA polymerase (RdRp) .

Structural modeling based on SARS-CoV-2 RdRp indicates that amino acids 228-267 (corresponding to this hotspot) are located near the pocket of RdRp and may be related to viral RNA binding . This positioning could explain why recombination in this region might confer replicative advantages to the virus.

While the provided research doesn't specifically identify recombination hotspots in the 2b/E coding region, the structural and functional studies of this protein contribute to our understanding of how recombination-driven changes in viral structural proteins might affect virus-host interactions and pathogenesis.

How do artificially constructed recombinant PRRSVs help study viral function?

Artificially constructed recombinant PRRSVs serve as powerful tools for investigating the functional significance of specific genomic regions and protein interactions. Research has demonstrated that:

• Recombinant PRRSVs harboring high-frequency recombination regions from different viral lineages can be constructed using infectious clone technology .

• Four recombinant PRRSVs were artificially constructed by switching complete or partial NSP9 (including the recombination hotspot) between two different PRRSV lineages (L1 PRRSV HeB108 and L8 PRRSV HuN4) .

• These artificially created recombinants showed increased viral genomic copies compared to their parental viruses, suggesting that dominant recombination patterns may be naturally selected to benefit viral replication .

• Using reverse genetics to create targeted mutations, such as the E gene knockout (P129-ΔE), allows precise determination of protein function in the viral life cycle .

This experimental approach provides mechanistic insights into why certain recombination patterns persist in nature and how specific genomic regions contribute to viral fitness.

How can researchers generate and validate E/2b protein-knockout PRRSV?

The generation and validation of E/2b protein-knockout PRRSV involves several key methodological steps:

• Mutation design in a full-length infectious clone:

  • The ATG translational initiation codon of the E gene is mutated to GTG

  • This targeted mutation prevents E protein synthesis while maintaining the overlapping ORF2a reading frame intact

• Transfection and validation:

  • The mutated genomic clone (P129-ΔE) is transfected into PRRSV-susceptible cells

  • Virion production is confirmed by electron microscopy and detection of viral genomic RNA in culture supernatant

  • The absence of infectivity is verified by attempting passage on fresh cells

• Functional analysis of E-deficient particles:

  • Strand-specific RT-PCR is performed to determine if E-negative particles can enter cells

  • This approach distinguishes between viral genomic RNA (negative strand) and mRNA (positive strand)

  • Detection of negative-strand RNA in cells exposed to E-deficient particles confirms virus entry but reveals a block in subsequent replication steps

This methodological approach definitively established that the E protein is dispensable for virion assembly but essential for infectivity, specifically at a post-entry stage of the viral life cycle.

What techniques are used to purify and characterize PRRSV virions for protein analysis?

The characterization of virion protein composition requires highly purified virus preparations. Researchers employ multiple complementary techniques:

• Two-step purification process:

  • Initial purification by sucrose-gradient centrifugation of labeled virus

  • Further purification by immunocapture using monoclonal antibodies against exposed virion proteins

  • This approach yields highly purified intact virions for protein analysis

• Metabolic labeling:

  • Infected cells are cultured with [³⁵S]-methionine/cysteine to radiolabel newly synthesized viral proteins

  • This enables sensitive detection of even minor virion components by autoradiography

• Immuno-detection methods:

  • Development of protein-specific monoclonal antibodies (like mAb 17C3 against the C-terminal peptide of 2b)

  • Use of these antibodies for Western blot and radioimmunoprecipitation (RIP) analysis

  • Competitive ELISA to confirm antibody specificity

• Electron microscopy:

  • Negative staining of purified virions for morphological characterization

  • Comparison of wild-type and mutant virus particles (like P129-ΔE) to assess structural integrity

The combination of these approaches provides robust evidence for the presence and stoichiometry of specific proteins within the virion structure.

How can researchers investigate the ion channel activity of viral proteins like 2b/E?

Several complementary approaches can be used to investigate the putative ion channel activity of viral proteins:

• Bacterial expression systems:

  • Expression of 2b/E protein in E. coli to assess growth arrest phenotypes

  • Measurement of membrane permeability changes in bacterial cells expressing the protein

• Pharmacological inhibition studies:

  • Treatment of virus-infected cells with known ion-channel blocking agents

  • Evaluation of specific stages of viral replication affected by these inhibitors

  • Determination of IC₅₀ values for different classes of ion channel blockers

• Cross-linking experiments:

  • Chemical cross-linking of proteins in infected cells or transfected cells

  • Analysis of oligomeric forms by SDS-PAGE and immunoblotting

  • Determination of the stoichiometry of protein complexes

• Planar lipid bilayer experiments:

  • Reconstitution of purified protein in artificial membranes

  • Electrophysiological measurements of ion conductance

  • This approach could further characterize the ion selectivity and gating properties of the channel

• Liposome permeabilization assays:

  • Incorporation of the protein into liposomes loaded with fluorescent dyes

  • Measurement of dye release as an indicator of membrane permeabilization

  • This approach can provide quantitative data on pore formation

While not all of these approaches were described in the provided research, they represent standard methodologies in the field for characterizing viral ion channels.

How does the 2b/E protein of PRRSV compare to similar proteins in other arteriviruses?

The 2b/E protein of PRRSV shows functional and structural similarities to analogous proteins in other members of the Arteriviridae family:

• Equine Arteritis Virus (EAV):

  • The E protein of EAV is considered functionally homologous to PRRSV 2b

  • Both are minor structural components recovered in small quantities from purified virions

  • EAV E has been better characterized in earlier studies

• Lactate Dehydrogenase-Elevating Virus (LDV):

  • Interestingly, Plagemann did not find evidence of an E-like protein in sucrose gradient-purified LDV, suggesting potential diversity in arterivirus structural composition

• Translational initiation differences:

  • Unlike the E protein of EAV, the translation of PRRSV 2b is initiated two nucleotides downstream of the ORF2a start codon

  • This specific translational arrangement is part of the bicistronic nature of mRNA2

The comparative analysis of these proteins across the arterivirus family provides evolutionary insights into their conserved functions and structural adaptations.

What research gaps remain in our understanding of PRRSV 2b/E protein?

Despite significant advances, several important questions about the PRRSV 2b/E protein remain unanswered:

• Detailed structural characterization:

  • High-resolution structural studies (X-ray crystallography or cryo-EM) of 2b/E oligomers would provide insights into channel formation

  • Membrane topology and orientation within the virion envelope need further clarification

• Ion selectivity and gating:

  • The specific ions transported by 2b/E channels have not been definitively characterized

  • Mechanisms regulating channel opening and closing during different stages of infection remain unknown

• Interaction partners:

  • Potential interactions between 2b/E and other viral or host proteins during virus assembly and uncoating

  • The possible association between 2b and the nucleocapsid (N) protein was suggested but requires confirmation

• Immunological significance:

  • The role of 2b/E as a target for protective immunity needs further investigation

  • Sera from PRRSV-infected pigs recognized the 2b peptide, suggesting immunogenicity

• Therapeutic targeting:

  • Development of specific inhibitors targeting 2b/E ion channel activity could represent a novel antiviral strategy

  • Optimization of broad-spectrum viroporin inhibitors for PRRSV-specific applications

Addressing these research gaps would significantly advance our understanding of PRRSV pathogenesis and potentially lead to new intervention strategies.