Recombinant Berne virus Truncated non-functional hemagglutinin-esterase homolog (HE)

What is the hemagglutinin-esterase (HE) protein in Berne virus and why is it considered truncated?

The hemagglutinin-esterase protein in Berne virus (also known as Equine Torovirus or EToV) is considered truncated because it contains a termination codon positioned in the middle of the gene, resulting in premature protein termination and loss of function . Unlike functional HE proteins found in related viruses isolated directly from infected animals, the truncated form in Berne virus cannot perform the dual functions of receptor binding (lectin activity) and receptor destruction (sialate-O-acetylesterase activity).

The truncation appears to be a consequence of adaptation to growth in cell culture conditions. The search results specifically note that "EToV Berne, which can multiply in cultured cells, also has an HE gene with a termination codon in the middle of the gene" . This characteristic is not unique to Berne virus but represents a common adaptation seen in multiple toroviruses and coronaviruses when passaged in laboratory cell lines.

Methodologically, researchers can verify this truncation through genetic sequencing of the HE gene region or through expression studies that demonstrate the absence of functional HE protein in Berne virus-infected cells.

How does cell culture passage affect HE expression in toroviruses and related viruses?

Cell culture passage exerts strong selective pressure on toroviruses and related coronaviruses, typically leading to truncation or elimination of functional HE protein expression. This phenomenon follows a consistent pattern across multiple virus types:

What methods can be used to detect and analyze the truncated HE protein in Berne virus-infected cells?

Detection and analysis of the truncated HE protein in Berne virus-infected cells requires specialized techniques due to the protein's altered structure and potentially reduced expression. Several methodological approaches have proven effective:

• PCR-based genetic detection: Researchers can use PCR with specific primers targeting the HE gene region. The search results describe primers such as "HE-for (5′ CCTAATAACTACTTAAAC 3′) and HE-rev (5′ GATGCTGAGTTTAATATTC 3′)" , which cover the initiation and termination codons, allowing amplification of the truncated gene.

• Expression systems with protein tags: Since detection of truncated HE can be challenging with standard antibodies, expression systems incorporating protein tags facilitate identification. The search results note: "Since the HE protein expressed in COS-7 cells failed to be detected by the anti-BToV antibody... we expressed HE protein with a tag at the C-terminal end" . This approach enables detection using antibodies against the tag rather than the viral protein itself.

• Immunofluorescence assays: Utilizing tag-specific antibodies in immunofluorescence assays allows visualization of the expression and cellular localization of recombinant HE proteins . This approach can confirm expression and provide insight into processing and trafficking.

• Functional hemagglutination assays: While the truncated HE lacks activity, hemagglutination (HA) assays can confirm this functional deficit. The search results indicate that "cells transfected with either the HE gene or the HE + tag gene showed robust HAD activity" when testing full-length HE, while truncated HE showed no activity.

Importantly, researchers should note that detection of truncated HE can be problematic: "The truncated HE protein was also expressed, but it did not exhibit HAD activity... the truncated HE protein was not detected" . This suggests the protein may be unstable, rapidly secreted, or expressed at levels below detection thresholds, requiring careful experimental design and multiple detection approaches.

What is the functional relationship between the spike protein (S) and HE in toroviruses, and how does this change with a truncated HE?

The functional relationship between the spike protein (S) and HE in toroviruses represents a sophisticated system of balanced activities that becomes disrupted when HE is truncated. This relationship can be characterized as follows:

• Complementary receptor interactions: In viruses with functional HE (like BCoV), S and HE work together in a coordinated manner where "S-mediated virion attachment and HE-mediated receptor destruction" create a dynamic balance . This allows virions to navigate through decoy receptors and locate true entry receptors.

• Functional interdependence: The search results explicitly state that "HE and S are functionally interdependent" , indicating that changes in one protein often necessitate compensatory changes in the other to maintain viral fitness.

• Disrupted balance with truncated HE: When HE is truncated (as in Berne virus), this balance is fundamentally altered. The search results describe how "destruction of the HE lectin RBS [receptor-binding site] creates an imbalance between S-mediated virion attachment and HE-mediated receptor destruction that can only be compensated by strongly reducing S affinity" .

• Molecular adaptation mechanisms: In the absence of functional HE, viruses adapt through "compensatory second-site mutations in S" that "dramatically reduce S RBS affinity, apparently to restore reversibility of binding and virion motility as an escape ticket from inadvertent virion attachment to decoy receptors" .

• Population-level compensatory strategies: Interestingly, viruses with truncated HE may develop complex population structures where "loss of HE lectin function was compensated at the population level" with some viruses maintaining high-affinity S proteins while relying on low-affinity variants to provide exogenous receptor-destroying activity .

In Berne virus with its naturally truncated HE, the S protein has likely adapted to function effectively without HE's complementary activities. This adaptation may involve modifications in S binding affinity, alterations in receptor specificity, or changes in the kinetics of S-mediated fusion to compensate for the loss of HE functionality.

What compensatory mutations occur in the spike protein when HE functionality is compromised?

When HE functionality is compromised, the spike protein undergoes specific compensatory mutations to maintain viral fitness and restore the balance between receptor binding and release. These adaptations show remarkable specificity:

• Location of mutations: Compensatory mutations cluster primarily in the receptor-binding domain (S1A). The search results describe that "single-amino acid mutations in receptor-binding domain S1A were limited to a finite number of positions. They were either within or proximal to the RBS to directly affect protein–ligand interactions, or more distal to reduce RBS affinity through long-range effects or by disrupting local folding through aberrant disulfide-bonding" .

• Specific mutations observed: Several specific mutations with quantified effects are documented:

  • Asn27Ser: Reduced S binding affinity to 9-O-Ac-Sia by approximately 500-fold

  • Ile26Ser: Reduced binding affinity by more than 10,000-fold

  • Gly82Glu: Reduced binding affinity by more than 10,000-fold

  • Thr83Ile, Thr83Asn, and Leu89Pro: Selected in response to HE-Thr114Asn substitution

• Dynamic evolutionary adaptation: The search results describe a fascinating process where "serial passage of the rBCoV-HE-Thr114Asn resulted in a succession of mutations alternatingly in HE and S" . This included reversion mutations (Thr83→Ile→Thr; Leu89→Pro→Leu) or near-reversions (Thr83→Ile→Ser; Leu89→Pro→Thr/Ser) as the virus continued to optimize the balance between binding and release.

• Functional consequences: These mutations "dramatically reduce S RBS affinity, apparently to restore reversibility of binding and virion motility as an escape ticket from inadvertent virion attachment to decoy receptors" . This demonstrates how viruses dynamically adjust receptor affinity to compensate for defects in receptor destruction capacity.

The pattern of these compensatory mutations reveals that viruses strive toward "optimal virion avidity" rather than simply maximizing binding strength, highlighting the sophisticated evolutionary mechanisms that maintain viral fitness when HE functionality is compromised.

How can recombinant Berne virus with restored HE functionality be developed and stabilized?

Developing recombinant Berne virus with restored HE functionality presents significant challenges due to the strong selective pressure against functional HE in cell culture. Based on the search results, several strategic approaches can be implemented:

• Reverse genetics systems: Researchers can employ targeted recombination approaches similar to those described for BCoV: "we performed targeted recombination and rescued recombinant viruses" . This would involve constructing a full-length cDNA clone of the Berne virus genome with a modified HE gene lacking the internal termination codon.

• Exogenous enzyme supplementation: The search results describe a crucial stabilization method using "exogenous soluble HE" added to the culture medium. Specifically, "concentrations of exogenous sialate-O-acetylesterase as low as 1 ng/mL to up to 1 μg/mL promoted virus growth" of recombinant viruses with functional HE. This approach could be essential for initial propagation of Berne virus variants with restored HE.

• Genetic stabilization: The search results demonstrate that when viruses were propagated with exogenous HE-Fc, "all viruses, cloned by endpoint dilution of the 160-h stock (n=4), coded for mutant HE-Phe211Ala in combination with wild-type S1A" . Further amplification maintained genetic stability, with NGS analysis showing "sequence variation in HE and S1A was distributed randomly and did not exceed background levels (<0.15%). More than 99.5% of the viruses coded for HE-Phe211Ala, while preserving parental type S1A" .

• Low-passage isolation: The search results suggest starting with fresh clinical isolates: "viruses with a lower passage history could have the full-length HE gene. We are, at the moment, trying to obtain BToV with a full-length HE protein from among the viruses with a shorter passage history in cultured cells" .

The key insight from these approaches is that successful stabilization of recombinant Berne virus with functional HE requires addressing the selective pressure that favors truncated variants. By providing exogenous receptor-destroying enzymes during propagation, researchers can maintain the genetic integrity of the recombinant virus while preventing the accumulation of compensatory mutations in the S protein.

How does the truncated HE affect viral replication kinetics and pathogenesis compared to viruses with functional HE?

The impact of truncated HE on viral replication kinetics and pathogenesis reveals the distinct requirements for viral fitness in different environments. Based on the search results, several key differences emerge:

• Enhanced replication efficiency in cell culture: Truncated HE appears to confer a replication advantage in cell culture systems. The search results explain that "the virus does not need to cope with the host defense mechanism and may discard the unnecessary component from virions, resulting in more efficient replication" . This suggests that Berne virus with its truncated HE is well-adapted for laboratory cultivation.

• Altered attachment-release dynamics: The balance between receptor binding and destruction is fundamentally changed with truncated HE. The search results emphasize that "in addition to an optimal balance between receptor binding and receptor destruction, the system strives toward optimal virion avidity" . In Berne virus, the truncated HE shifts this balance, affecting how virions attach to and release from cellular receptors.

• Reduced pathogenic potential in vivo: The search results indicate that while HE is "not indispensable for virus replication in cultured cells," it likely "may play an important role in the pathogenesis of diarrhea caused by this virus" . This suggests that Berne virus with truncated HE may exhibit attenuated virulence in natural host environments compared to viruses with functional HE.

• Host barrier navigation deficiencies: Functional HE appears important for overcoming host defenses: "Possibly, the esterase activity of the HE protein has the ability to destroy mucus, thus making it easier for the virus to attach to the receptor of epithelial cells lacking a mucus coat" . Berne virus with truncated HE may therefore show impaired ability to navigate mucosal barriers in vivo.

• Dependency relationships in mixed populations: In mixed viral populations, variants with different HE functionalities can establish cooperative relationships. The search results describe how viruses with low-affinity S proteins can provide "aid by serving as a source of exogenous sialate-O-acetylesterase activity" for high-affinity variants.

These findings illustrate how truncated HE in Berne virus represents an adaptation to laboratory conditions that likely comes at the cost of reduced fitness in natural host environments. This trade-off between in vitro replication efficiency and in vivo pathogenic potential offers important insights into viral evolution and adaptation.

How can researchers differentiate between phenotypic effects caused by HE truncation versus other viral factors?

Differentiating phenotypic effects specifically attributable to HE truncation from those caused by other viral factors requires systematic experimental approaches. Based on the search results, several methodological strategies can be implemented:

• Isogenic recombinant virus systems: Creating recombinant viruses that differ only in HE functionality enables direct comparison of phenotypic effects. The search results describe such an approach: "To better understand the consequences of loss of HE lectin function as it occurred during OC43 and also HKU1 evolution, we took a reverse genetics/forced evolution approach with BCoV as a model" .

• Trans-complementation studies: Providing functional HE in trans can determine if observed phenotypes are directly related to HE deficiency. The search results describe adding "exogenous soluble HE to the culture medium" at concentrations ranging from "1 ng/mL to up to 1 μg/mL" to restore growth of HE-deficient viruses, demonstrating that growth deficiency was specifically due to HE dysfunction.

• Sequential passage analysis: Monitoring viral adaptation through sequential passages can reveal the relationship between HE function and other viral components. The search results describe how "serial passage of the rBCoV-HE-Thr114Asn resulted in a succession of mutations alternatingly in HE and S" , demonstrating the functional interrelationship between these proteins.

• Site-directed mutagenesis: Creating specific mutations in HE while maintaining other viral components constant helps isolate HE-specific effects. The search results describe experiments with "destruction of the HE lectin RBS" through specific mutations like "HE-Phe211Ala" .

• Population-level genomic analysis: Next-generation sequencing analysis of viral populations can distinguish between HE-related adaptations and other evolutionary changes. The search results mention using NGS to analyze viral populations, which "allows for the detection of low-frequency mutants" .

By systematically applying these approaches, researchers can establish causal relationships between HE truncation and specific phenotypic changes in Berne virus or related toroviruses, advancing our understanding of the biological significance of this protein in different viral life cycle stages and host environments.