Recombinant Varicella-zoster virus Ribonucleoside-diphosphate reductase small chain (ORF18)

What is the function of Ribonucleotide Reductase in Varicella-zoster virus replication?

Ribonucleotide reductase (RR) in VZV catalyzes the conversion of ribonucleotides to deoxyribonucleotides, providing the building blocks necessary for viral DNA synthesis. The enzyme consists of two subunits: the large catalytic subunit encoded by ORF19 and the small regulatory subunit encoded by ORF18.

Unlike cellular ribonucleotide reductase, the viral enzyme is not allosterically inhibited by its metabolites, leading to higher intracellular dNTP pools during infection . This characteristic affects the virus's susceptibility to nucleoside analogs like acyclovir. Studies have shown that cells infected with VZV express a viral ribonucleotide reductase distinct from that present in uninfected cells, and deletion of the large subunit (ORF19) impairs viral growth in vitro and increases susceptibility to acyclovir .

Methodology for studying RR function typically involves:

• Enzyme activity assays using radiolabeled substrates (e.g., [³H]CDP)

• Viral growth kinetics in the presence of RR inhibitors

• Comparison of viral replication efficiency between wild-type and RR-deficient recombinant viruses

How is ORF18 expression regulated during the VZV replication cycle?

VZV gene expression follows a cascade-like temporal pattern similar to other herpesviruses, with genes classified as immediate-early (IE), early (E), or late (L). ORF18, encoding the small subunit of ribonucleotide reductase, is generally considered an early gene based on its function in nucleotide metabolism preceding viral DNA synthesis.

Recent comprehensive transcriptome analysis has provided insight into the kinetics of VZV gene expression. Interestingly, some genes previously classified based on homology to HSV-1 have shown different expression patterns in VZV. For example, ORF62, which encodes the major viral transactivator protein and was expected to be expressed with immediate-early kinetics (like its HSV-1 ortholog), was found to be expressed with late kinetics in VZV .

Methodological approaches to study ORF18 expression kinetics include:

• Time-course analysis using qPCR to quantify ORF18 transcript levels at different time points post-infection

• Western blotting with specific antibodies to detect protein expression over time

• Using metabolic inhibitors (e.g., cycloheximide, phosphonoacetic acid) to block different phases of viral replication and assess effects on ORF18 expression

• Reporter viruses with fluorescent tags to visualize expression patterns

How do VZV ORF18 and ORF19 interact to form functional ribonucleotide reductase?

The functional VZV ribonucleotide reductase enzyme requires both the small (ORF18) and large (ORF19) subunits. Research indicates that deletion of ORF19 alone abolishes viral ribonucleotide reductase activity, demonstrating the essential role of both subunits in forming a functional enzyme complex .

When testing viral ribonucleotide reductase activity in cell lysates from VZV ROka 19D (ORF19 deletion mutant), researchers observed only 1.7% conversion of CDP to dCDP, similar to uninfected cells (1.6% conversion). In contrast, lysates from cells infected with parental ROka exhibited 17% conversion, indicating substantial ribonucleotide reductase activity .

Methodological approaches to studying subunit interactions include:

• Co-immunoprecipitation assays with tagged ORF18 and ORF19

• Yeast two-hybrid or mammalian two-hybrid assays

• Bimolecular fluorescence complementation (BiFC)

• Förster resonance energy transfer (FRET) with fluorescently labeled subunits

• X-ray crystallography or cryo-EM to determine the structure of the enzyme complex

What cell culture systems are suitable for studying VZV ORF18 function?

Several cell culture systems have been used to study VZV gene functions, including ORF18:

SH-SY5Y neuroblastoma cells have been used to study VZV gene expression during latency and reactivation. These cells can be differentiated into neuron-like cells and infected with low-titer cell-free VZV in the presence of acyclovir to establish a quiescent infection .

Human embryonic stem cell-derived neurons provide a more physiologically relevant system for studying VZV neurotropism and can host a persistent non-productive infection that can be reactivated by experimental stimuli .

What methods are used to create recombinant VZV with ORF18 modifications?

Several genetic engineering approaches have been developed to create recombinant VZV strains:

• Cosmid-based mutagenesis: This involves dividing the VZV genome into four overlapping cosmids. Mutations are introduced into the relevant cosmid (containing ORF18), and all four cosmids are then co-transfected into cells to reconstitute the virus .

• Bacterial Artificial Chromosome (BAC) system: The entire VZV genome is maintained as a BAC in E. coli, allowing for more efficient genetic manipulation. The BAC-based approach has been used for both the vaccine Oka strain and laboratory strains like Ellen .

Methodology for BAC mutagenesis of ORF18:

• The recombinant VZV-BAC episome is extracted from infected cells (e.g., ARPE-19)

• The BAC is transferred into bacterial cells expressing the Lambda Red recombineering system (e.g., SW102)

• ORF18 modifications are introduced through homologous recombination

• The modified BAC is transfected back into mammalian cells for virus reconstitution

For example, researchers have successfully constructed VZV BAC vectors based on the vaccine strain vOka and the laboratory strain Ellen, inserting reporter genes between specific ORFs and introducing mutations to study protein function .

How does deletion or mutation of ORF18 affect VZV replication compared to ORF19 deletion?

ORF19 deletion effects include:

• Viral ribonucleotide reductase activity is abolished

• Smaller plaque size (0.34 ± 0.10 mm vs. 1.1 ± 0.17 mm for wild-type)

• Slower growth kinetics after day 3 post-infection

• Increased sensitivity to acyclovir (IC50 of 1.5-1.8 μg/ml vs. 4.9 μg/ml for wild-type)

Methodological approaches to compare ORF18 and ORF19 deletion effects:

• Growth curves in different cell types and culture conditions

• Plaque size comparisons

• Acyclovir sensitivity assays

• Metabolomic analysis of dNTP pools

• In vivo pathogenesis studies using humanized mouse models

What is the potential of ORF18 as a target for antiviral therapies?

Ribonucleotide reductase represents an attractive target for antiviral therapy for several reasons:

• Synergy with nucleoside analogs: Inhibition of viral ribonucleotide reductase enhances the efficacy of nucleoside analogs like acyclovir by increasing the ratio of acyclovir triphosphate to dGTP inside infected cells .

• Differential inhibition potential: The structural differences between viral and cellular ribonucleotide reductases may allow for selective targeting of the viral enzyme.

• Attenuated virulence: As demonstrated with ORF19 deletion, inhibition of ribonucleotide reductase impairs viral replication without completely preventing it, suggesting a potentially favorable therapeutic window.

Compound 348U87, a ribonucleotide reductase inhibitor, has been shown to potentiate the activity of acyclovir against VZV similarly to the effect seen with ribonucleotide reductase deletion mutants .

Methodological approaches for developing ORF18-targeted antivirals:

• High-throughput screening of compound libraries against recombinant ORF18/ORF19 complex

• Structure-based drug design based on crystal structures

• Peptide inhibitors targeting the interface between ORF18 and ORF19

• Evaluation of drug combinations (ribonucleotide reductase inhibitors plus nucleoside analogs)

• Development of ORF18-specific small interfering RNAs (siRNAs) or antisense oligonucleotides

How can ORF18 be utilized in the development of attenuated VZV vaccines?

Modifications to ribonucleotide reductase genes have potential applications in vaccine development:

• Attenuated replication: Mutations in ORF18 could produce viruses with attenuated replication capability while maintaining immunogenicity.

• Increased sensitivity to antivirals: As seen with ORF19 deletion, modifying ribonucleotide reductase increases sensitivity to acyclovir, providing an additional safety feature for live attenuated vaccines.

• Vector for heterologous antigens: Recombinant VZV with modifications to ORF18 could serve as a vector for expressing antigens from other pathogens. VZV has been used to express foreign viral genes from herpes simplex, Epstein-Barr virus, hepatitis B, mumps, HIV, and simian immunodeficiency virus .

Historical perspective: The current VZV vaccine strain (Oka) was empirically attenuated through serial passage in cell culture. More rational approaches to attenuation, such as targeted modifications to ORF18, could lead to improved vaccine candidates with better safety profiles or enhanced immunogenicity.

Research indicates that ribonucleotide reductase-deficient HSV-1 can establish latency in the central nervous system of mice but cannot reactivate . Similarly, ribonucleotide reductase deletion mutants of pseudorabies virus are avirulent and elicit protective immunity in pigs. These observations suggest that VZV strain Oka with ribonucleotide reductase modifications could potentially be a superior live virus vaccine candidate .

How does ORF18 expression vary between lytic infection and latency in VZV?

Understanding the expression patterns of ORF18 across different phases of the viral life cycle provides insights into its role in VZV pathogenesis:

During lytic infection, ORF18, encoding the small subunit of ribonucleotide reductase, is expected to be expressed as an early gene to support viral DNA replication. During latency, viral gene expression is highly restricted, with only a limited set of transcripts detected.

In latently infected human trigeminal ganglia, the predominant transcripts are the VZV latency-associated transcript (VLT) and associated VLT-ORF63 splice variants . Recent research has also identified circular RNAs (circRNAs) during VZV infection, including VZV latency-associated transcript-like circRNAs (circVLTs) .

Methodological approaches to study ORF18 expression during latency:

• Single-cell RNA sequencing of latently infected neurons

• In situ hybridization in human ganglia specimens

• Analysis of chromatin modifications at the ORF18 promoter during latency

• Use of reporter viruses in latency models

• Comparison of ORF18 expression in in vitro latency models with different stimuli

The SH-SY5Y neuroblastoma cell line has been used as a model to investigate VZV latency. When these cells are differentiated into neuron-like cells and infected with low-titer VZV in the presence of acyclovir, a quiescent infection can be established . This system could be useful for studying ORF18 expression during the transition between lytic and latent states.

What is the role of ORF18 in oncolytic VZV development?

Oncolytic virotherapy using engineered VZV represents an emerging area of research. The potential role of ORF18 modifications in this context includes:

• Attenuation for safety: Mutations in ribonucleotide reductase genes could provide a mechanism to attenuate viral replication in normal tissues while maintaining oncolytic activity in cancer cells, which often have dysregulated nucleotide metabolism.

• Tumor selectivity: Cancer cells frequently have elevated dNTP pools due to upregulated nucleotide metabolism, potentially complementing deficiencies in viral ribonucleotide reductase function.

• Compatibility with immune-stimulatory modifications: As demonstrated with Ellen-ΔORF8-tet-off-scIL12, a VZV vector with deleted deoxyuridine triphosphatase (ORF8) and an inducible IL-12 expression cassette, attenuation of specific viral functions can be combined with immunostimulatory modifications to enhance therapeutic efficacy .

Recent research has explored the oncolytic potential of VZV:

• VZV strains vOka and Ellen exhibited potent antitumor efficacy in a MeWo melanoma xenograft model

• Deletion of ORF8 (encoding viral deoxyuridine triphosphatase) attenuated VZV replication both in vitro and in vivo without compromising oncolytic potency

• Addition of an inducible mouse single-chain IL-12 (scIL12) gene cassette to the attenuated vector triggered systemic antitumor immune responses in an immunocompetent melanoma model

Similar approaches could be applied to ORF18 to develop novel oncolytic VZV vectors with enhanced safety and efficacy profiles.