Recombinant Crimean-Congo hemorrhagic fever virus RNA-directed RNA polymerase L (L), partial

What is the CCHFV L protein and what functional domains does it contain?

The CCHFV L protein is an unusually large multifunctional protein (approximately 450 kDa) that serves as the viral RNA-dependent RNA polymerase (RdRp) . This essential viral enzyme catalyzes both replication of the viral genome and transcription of viral mRNAs. The L protein contains multiple functional domains, including:

• N-terminal ovarian tumor-type protease (OTU) domain that exhibits deubiquitinating (DUB) activity

• RdRp domain with conserved polymerase motifs

• Additional domains with predicted functions in cap-snatching and endonuclease activities

The OTU domain (approximately 200 amino acids) cleaves both ubiquitin and ISG15 modifiers from target proteins, enabling the virus to interfere with innate immune responses . The RdRp domain contains conserved motifs found in other viral polymerases and is responsible for synthesizing viral RNA through primer-dependent and de novo mechanisms . Mutations in the putative active site, such as D2517N, render the enzyme inactive, confirming its essential role in viral replication .

What methodologies are used to express and purify recombinant CCHFV L protein?

Recombinant full-length CCHFV L protein has been successfully expressed in insect cell expression systems and purified to near homogeneity using affinity chromatography techniques . The specific methodology includes:

• Cloning the L gene into baculovirus expression vectors

• Transfection and infection of insect cells (typically Sf9 or High Five cells)

• Expression optimization through temperature, time, and multiplicity of infection adjustments

• Affinity purification using tags such as His-tag or FLAG-tag

• Further purification through size exclusion chromatography or ion exchange chromatography

This approach yields functionally active L protein capable of both RNA synthesis and deubiquitinating activities that can be used for biochemical characterization and inhibitor screening . The purified enzyme demonstrates activity in the presence of divalent metal ions, producing full-length RNA products as well as shorter products when specific nucleotides are omitted from reaction mixtures .

How are recombinant CCHFV systems developed and what are their research applications?

Reverse genetics systems have been developed to produce recombinant CCHFV, overcoming a major impediment in CCHFV research . These systems typically involve:

• Construction of plasmids encoding the viral S, M, and L genome segments

• Co-transfection of these plasmids into permissive cell lines (such as BSR-T7/5 or Huh7 cells)

• Expression of viral RNA segments using bacteriophage T7 RNA polymerase

• Trans-support with CCHFV nucleoprotein and L polymerase for encapsidation

• Recovery and amplification of recombinant viruses

The systems have been optimized to systematically recover high yields of infectious CCHFV and enable the production of specifically designed CCHFV mutants . Research applications include:

• Structure-function studies of viral proteins

• Investigation of viral replication mechanisms

• Design and testing of attenuated vaccine candidates

• Studying host-pathogen interactions

• Screening of antiviral compounds

For example, researchers have used these systems to investigate the role of furin cleavage in CCHFV glycoprotein maturation by generating recombinant viruses with mutations in the furin cleavage motif .

What are the challenges in structural elucidation of CCHFV L protein and how are they being addressed?

The crystal structure of CCHFV L protein remains unresolved due to several challenges:

• The large size (~450 kDa) makes crystallization difficult

• Multi-domain architecture with potential flexible regions

• Technical difficulties in expressing and purifying sufficient quantities of stable protein

• Biosafety level 4 (BSL-4) containment requirements for working with infectious CCHFV

These challenges are being addressed through in silico approaches to structural modeling . The methodology includes:

• Identification of appropriate template structures with functional similarity despite low sequence identity

• Stepwise homology modeling with careful template selection

• Iterative refinement through molecular dynamics (MD) simulations (typically 20-100 ns)

• Model validation using Ramachandran plots and MolProbity metrics

• Selection of representative conformations from MD trajectory clusters based on RMSD cutoffs

How do nucleotide analogs and other compounds inhibit CCHFV L protein activity?

Inhibition of CCHFV L protein has been studied using various compounds, with different mechanisms of action observed :

• Nucleoside/nucleotide analogs: These compounds mimic natural nucleotide substrates and act as chain terminators or induce mutations through base mispairing.

  • Ribavirin and favipiravir triphosphate forms compete with ATP or GTP but are incorporated less efficiently than natural nucleotides

  • 2'-deoxy-2'-fluoro-CTP (FdC) and 2'-deoxy-2'-amino-CTP show increased inhibitory effects due to higher rates of incorporation compared to ribavirin and favipiravir

• Non-nucleoside inhibitors: These compounds may bind to allosteric sites and interfere with polymerase function.

The inhibition mechanism involves:

• Competition with natural nucleotide substrates

• Chain termination after incorporation

• Induction of lethal mutagenesis

• Disruption of critical protein-protein or protein-RNA interactions

Research methodologies to evaluate inhibitors include:

• In vitro polymerase assays using purified recombinant L protein

• Measurement of RNA synthesis using labeled nucleotides

• Assessment of inhibitor incorporation rates compared to natural nucleotides

• Structure-based design of inhibitors using in silico models

• Cell-based antiviral assays using recombinant viruses or replicon systems

What is the relationship between the OTU domain and ISG15 in CCHFV replication?

The relationship between the OTU domain of CCHFV L protein and ISG15 (Interferon-Stimulated Gene 15) reveals complex interactions essential for viral replication :

• The OTU domain cleaves both ubiquitin and ISG15 modifiers from target proteins

• This activity is thought to counteract cellular antiviral responses by preventing ISGylation of proteins involved in innate immunity

• Unexpectedly, the OTU domain also plays a critical role in regulating the activity of the L protein itself

Studies using transcriptionally active virus-like particles (tc-VLPs) have demonstrated that:

• The C40A mutation in the OTU domain renders it catalytically inactive

• This mutation attenuates CCHFV polymerase activity in human cells

• The attenuation cannot be relieved by inactivating the IFN response

• Overexpression of conjugation-competent ISG15 recovers the polymerase activity to wild-type levels

These findings suggest a cis requirement of the OTU protease for optimal CCHFV polymerase activity, specifically in relation to ISG15 . Rather than merely antagonizing host antiviral responses, the OTU domain appears to regulate CCHFV polymerase function through ISG15-dependent mechanisms. This dual role makes the OTU domain a potential target for antiviral strategies, and OTU-deficient tc-VLPs have been proposed as potential vaccine candidates .

What diagnostic approaches utilize recombinant CCHFV L protein components?

Recombinant CCHFV proteins, including components derived from the L protein, have contributed to the development of improved diagnostic methodologies :

• Recombinant ELISA (Rec-ELISA): While primarily utilizing nucleocapsid (NP) and mucin-like domain (MLD) proteins rather than L protein components, these assays demonstrate the utility of recombinant viral proteins in diagnostics. They show high sensitivity (97%) and specificity (98%) for convalescent cases .

• Recombinase Polymerase Amplification (RPA): This isothermal amplification technique targets viral genomic segments, including the L segment, for rapid molecular detection:

  • Enables detection under 10 minutes

  • Shows high target specificity across all 7 S-segment clades

  • Tolerates inhibitors in crude preparations of field samples

  • Can be performed on portable, lightweight real-time detection devices

  • Particularly valuable for field diagnostics in remote regions or low-resource laboratories

The RPA methodology involves:

• Target-specific primers and probes designed against conserved regions

• Isothermal amplification at 37-42°C (no thermal cycling required)

• Use of recombinase enzymes to facilitate strand invasion

• Integration with lateral flow strips or real-time fluorescence detection

• Minimal sample preparation requirements

This approach has been successfully validated with clinical samples from endemic regions such as Tajikistan, demonstrating its potential for point-of-need monitoring during CCHF outbreaks .

What are the main hurdles in developing effective therapies targeting CCHFV L protein?

Despite significant research progress, several challenges remain in developing effective therapies targeting the CCHFV L protein:

Future research directions should focus on:

• Structure determination through cryo-electron microscopy

• Fragment-based drug discovery approaches

• Allosteric inhibitors that can simultaneously affect multiple functions

• Host-directed therapies targeting essential L protein-host factor interactions

• Combination therapies targeting different viral processes

How can reverse genetics systems be optimized for CCHFV vaccine development?

Reverse genetics systems provide powerful platforms for rational vaccine design, with several optimization strategies for CCHFV vaccine development :

• Systematic optimization of plasmid ratios: Transfection conditions significantly impact virus recovery. Optimizing the ratio of S, M, and L segment-expressing plasmids, along with helper plasmids, can enhance rescue efficiency.

• Cell line selection and modification: Different cell lines support varying levels of CCHFV replication. Engineering cell lines to express optimal levels of host factors required for efficient virus rescue can improve yields.

• Targeted attenuation strategies:

  • OTU domain mutations: Since OTU-deficient tc-VLPs have been proposed as vaccine candidates, introducing specific mutations in the OTU domain can generate attenuated viruses that still induce protective immunity .

  • Glycoprotein processing mutations: Modifications in furin cleavage sites can alter glycoprotein maturation, potentially attenuating the virus while maintaining immunogenicity .

  • Codon deoptimization: Systematic replacement of codons with less efficiently translated alternatives can decrease viral protein expression and attenuate replication.

• Temperature-sensitive mutations: Engineering mutations that restrict viral replication at higher temperatures can create attenuated strains that replicate efficiently in vitro but are attenuated in vivo.

• Reporter gene incorporation: Insertion of reporter genes allows for easy monitoring of viral replication and spread, facilitating screening of antiviral compounds and vaccine efficacy.

Implementation of these strategies requires:

• Thorough characterization of attenuated phenotypes

• Evaluation of genetic stability of attenuated strains

• Assessment of immunogenicity and protective efficacy in animal models

• Safety testing to ensure lack of reversion to virulence