Recombinant Yaba monkey tumor virus Transcript termination protein A18 (110R)

What is Yaba Monkey Tumor Virus (YMTV) and how is it classified taxonomically?

YMTV is a member of the Yatapoxvirus genus of poxviruses that causes a distinctive disease in primates characterized by epidermal histiocytomas primarily affecting the head and limbs . It belongs to the Chordopoxvirinae subfamily, which includes other significant poxviruses such as Variola virus and Molluscum contagiosum virus . The virus was first identified in captive rhesus monkeys but has since been detected in other primates including vervet monkeys in South Africa . Taxonomically, YMTV shares genetic similarities with other yatapoxviruses such as tanapox virus, with key distinguishing genes related to host immune evasion.

What are the key functional domains of the YMTV A18 transcript termination protein?

The A18 protein in poxviruses functions as a transcript termination factor with DNA-dependent ATPase and helicase activities. While the search results don't provide specific information about YMTV A18's domains, comparative analysis with orthologous proteins in related poxviruses indicates it likely contains:

• An N-terminal ATPase domain for energy coupling

• A central helicase domain for unwinding DNA-RNA hybrids

• C-terminal regions that interact with other viral transcription factors

• Conserved motifs involved in nucleic acid binding

These domains work in concert to regulate the termination of intermediate and late viral gene transcription, a process essential for the temporal control of poxvirus gene expression during infection.

How does YMTV's genome structure compare to other poxviruses?

YMTV possesses a linear double-stranded DNA genome with characteristic terminal hairpin loops and inverted terminal repeats typical of poxviruses. Molecular analysis of the YMTV genome has been performed by amplifying regions such as the insulin metalloprotease-like protein, intracellular mature virion (IMV) membrane protein genes, and DNA polymerase gene . Phylogenetic analyses of these regions revealed that YMTV isolates typically show ~99% sequence similarity to reference strains .

The genome encodes proteins involved in various aspects of the viral life cycle, including the 14L protein that functions as an IL-18 binding protein to inhibit host immune responses . Unlike some other poxviruses, YMTV has evolved specific adaptations for primate hosts, including mechanisms to evade human immune responses, suggesting its potential reservoir may include non-human primates.

What are the optimal conditions for expressing recombinant YMTV A18 protein in laboratory settings?

Based on approaches used for other YMTV proteins, such as the 14L protein described in the search results, a recombinant baculovirus expression system offers significant advantages for producing YMTV A18. The methodology involves:

• PCR amplification of the A18 (110R) gene from YMTV genomic DNA

• Cloning into a suitable expression vector (e.g., pFastbac 1)

• Generation of recombinant baculovirus using the Bac-to-Bac system

• Expression in insect cells (e.g., Sf9 or High Five cells)

• Purification via affinity chromatography

For optimal expression, consider replacing the native signal sequence with an efficiently secreted signal peptide (such as the M-T7 signal sequence from myxoma virus) to enhance protein secretion, as demonstrated successfully with YMTV 14L protein . Expression should be performed at 27-28°C for 72 hours post-infection, followed by purification using either His-tag or other affinity methods.

What techniques are most effective for studying protein-protein interactions involving YMTV A18?

Several complementary techniques are recommended for investigating interactions between YMTV A18 and potential binding partners:

• Surface Plasmon Resonance (SPR): Immobilize purified A18 protein to a CM5 chip and analyze binding kinetics with potential interaction partners. This approach was successfully used to characterize YMTV 14L interactions with IL-18, revealing binding affinities in the nanomolar range .

• Co-immunoprecipitation assays: Using tagged versions of A18 (such as Myc/His-tagged constructs) and protein A/G beads pre-absorbed with specific antibodies to pull down protein complexes from infected cells .

• Functional sequestration assays: Similar to those used for YMTV 14L protein, these can determine if A18 can physically bind and sequester its target proteins .

• Mutagenesis studies: Generate alanine substitution mutants of key residues to map interaction sites, similar to approaches used for mapping IL-18 binding to YMTV 14L .

These methods can be combined with structural analyses to comprehensively characterize the protein interaction network of YMTV A18.

What molecular detection methods can identify YMTV in clinical or research samples?

For reliable detection of YMTV in biological samples, a comprehensive molecular approach is recommended:

• DNA extraction: Isolate viral DNA using appropriate tissue extraction kits (e.g., DNeasy Tissue MiniPrep kit) .

• PCR amplification: Target multiple conserved gene regions including:

  • 220 bp region of the insulin metalloprotease-like protein

  • Intracellular mature virion (IMV) membrane protein genes

  • 1082 bp fragment of the DNA polymerase gene

• Sequencing and phylogenetic analysis: Sequence PCR products and perform comparative analysis with reference sequences. Successfully amplified fragments should produce readable sequences of approximately 190-213 bp (IMV) and 1005-1020 bp (DNA polymerase) .

• Real-time PCR: For quantitative detection, develop specific primers for conserved regions unique to YMTV.

This multi-target approach increases specificity and sensitivity, reducing the risk of false negatives in samples with low viral loads.

How does the transcript termination mechanism of YMTV A18 differ from other poxviral termination factors?

Poxviral transcript termination mechanisms involve complex interactions between multiple viral proteins, including the A18 protein which functions as an RNA helicase. While specific details about YMTV A18 are not provided in the search results, comparative analysis with other poxviruses suggests:

• A18 likely works in conjunction with other viral factors to recognize specific termination signals

• It may possess ATPase activity to provide energy for the termination process

• The protein could interact with the viral RNA polymerase complex to mediate termination

In vaccinia virus, mutations in DNA polymerase genes can affect genetic recombination through single-strand annealing reactions (SSA), suggesting potential interplay between replication and transcription machinery . A similar mechanism might operate in YMTV, where A18 could influence both transcription termination and recombination events during viral replication.

What evolutionary adaptations in YMTV A18 contribute to host specificity?

While the search results don't directly address YMTV A18 host adaptations, insights can be drawn from evolutionary patterns observed in other YMTV proteins:

The 14L protein of YMTV has evolved to bind both human and murine IL-18 with high affinity (4.1 nM and 6.5 nM, respectively) . This adaptation likely represents an evolutionary strategy to enhance virus fitness in different host environments. Similarly, A18 may have evolved specific adaptations that optimize its function in primate hosts.

The binding specificity of YMTV 14L differs from that of Variola virus IL-18BP, interacting with two distinct clusters of IL-18 surface residues rather than a single cluster of three amino acids . This altered binding specificity demonstrates the plasticity of poxviral inhibitor domains and suggests A18 might similarly display host-specific adaptations.

Such evolutionary modifications in viral regulatory proteins like A18 may contribute to YMTV's ability to infect specific primate hosts while maintaining the potential to cross species barriers.

How do nucleotide pool dynamics influence YMTV A18 function during infection?

The search results indicate that poxviral ribonucleotide reductase (RR) small subunits can form functional complexes with host large RR subunits to provide sufficient nucleotide pools to support DNA replication . This suggests a model where local nucleotide concentrations near replication forks could influence the activities of various viral proteins, including potentially A18.

The interaction of viral single-stranded DNA-binding (SSB) proteins and RR at replication forks can modulate DNA polymerase activity through changes in local nucleotide pools . By extension, these dynamics may also affect A18 activity, as transcription termination is tightly coupled to replication in poxviruses.

A proposed model suggests that nucleotide pool modulation serves to "maximize replication rates while still allowing for recombinational repair" . This balance may be critical for A18 function, as proper transcript termination is essential for coordinated gene expression during the viral life cycle.

What are the key considerations when designing inhibitors targeting YMTV A18 protein?

When designing inhibitors against YMTV A18, researchers should consider:

• Structural analysis: While specific structural information about YMTV A18 isn't provided in the search results, researchers should use homology modeling based on related poxviral proteins to identify potential binding pockets. The approach used to map mutations in Vaccinia virus DNA polymerase onto the RB69 DNA polymerase structure illustrates this comparative structural approach .

• Functional domains: Target conserved functional domains such as the presumed ATPase and helicase regions, which are likely essential for A18 activity.

• Selectivity assessment: Design inhibitors that selectively target viral rather than host proteins to minimize toxicity, similar to how cidofovir derivatives (ANP, CDV) target viral DNA polymerases .

• Resistance profiling: Consider the potential for resistance development. Studies of ANP-resistant Vaccinia strains revealed specific point mutations in the DNA polymerase gene that conferred resistance . Similar resistance mechanisms might develop against A18 inhibitors.

• In vivo testing: Assess attenuation in animal models, as drug-resistant poxvirus strains may show reduced pathogenicity in vivo despite robust replication in culture .

What cell culture systems are most appropriate for studying YMTV A18 function?

Based on the search results and knowledge of poxvirus research, the following cell culture systems are recommended:

• Primate cell lines: CV-1 cells (derived from African green monkey kidney) are suitable for YMTV propagation, as demonstrated by growing YMTV (VR587) at 34°C on CV1 cells .

• Human cell lines: For studying interactions with human host factors, human cell lines such as KG-1 cells can be used to measure functional outcomes, as demonstrated in IL-18 bioactivity assays .

• Reporter systems: Develop reporter constructs to monitor A18-dependent transcript termination efficiency in infected cells.

• Temperature considerations: Maintain cultures at 34°C rather than 37°C for optimal YMTV replication, as this lower temperature better reflects the virus's natural conditions .

• BSL-2+ containment: Given that YMTV can potentially infect humans, all experiments should be conducted under appropriate biosafety conditions.

How can CRISPR-Cas9 technology be applied to study YMTV A18 function in viral replication?

CRISPR-Cas9 technology offers powerful approaches for investigating YMTV A18 function:

These approaches can help elucidate the role of A18 in YMTV replication and potentially identify novel targets for antiviral intervention.

How should researchers interpret conflicting results between in vitro binding assays and functional inhibition studies?

The search results provide an excellent case study for this question through the YMTV 14L protein research, which demonstrated a discrepancy between binding and functional inhibition:

To interpret such discrepancies, researchers should:

• Consider conformational heterogeneity: The authors hypothesized that "a fraction of the IL-18 protein was in a state or conformation that was not functionally inhibited even when bound to 14L" .

• Evaluate assay conditions: Different buffer conditions, temperatures, or protein concentrations between assays may affect binding vs. function.

• Examine competitive factors: In cellular assays, competition with endogenous factors may occur that doesn't exist in purified protein binding studies.

• Design alternative assays: As demonstrated with the YMTV 14L study, creating a third assay (the sequestration assay) helped reconcile the conflicting results from binding and functional studies .

• Assess biological relevance: Determine which assay system most closely mimics in vivo conditions.

What statistical approaches are most appropriate for analyzing viral protein-host interaction data?

When analyzing viral protein-host interaction data, researchers should consider these statistical approaches:

• Binding kinetics analysis: For surface plasmon resonance data, use appropriate curve-fitting models to determine kon and koff rates and calculate equilibrium dissociation constants (KD). The search results show this approach was used for analyzing YMTV 14L binding to IL-18 .

• Dose-response analysis: For functional inhibition assays, calculate IC50 values and use appropriate regression models (linear, logarithmic, or sigmoidal) based on the biological system. The YMTV 14L inhibition of IFN-γ production was analyzed using dose-dependent curves .

• Multiple independent experiments: Ensure reproducibility by performing experiments with both tagged and untagged versions of proteins, as done with YMTV 14L .

• Controls: Include appropriate positive and negative controls. For YMTV 14L studies, controls included human IL-18BP, neutralizing antibody to IL-18, and IL-18 receptor blocking antibody .

• Multivariate analysis: For complex datasets involving multiple mutations or conditions, principal component analysis or cluster analysis may reveal patterns not evident in univariate statistics.

• Phylogenetic analysis: When comparing sequences across viral species, appropriate evolutionary models should be selected for sequence alignment and tree construction, as demonstrated in the molecular detection of YMTV .

How can researchers distinguish between direct and indirect effects of A18 dysfunction on viral replication?

Distinguishing direct from indirect effects of A18 dysfunction requires specialized experimental approaches:

• Temporal expression analysis: Monitor transcript and protein levels at different timepoints after infection to determine if A18 dysfunction directly affects specific viral gene expression patterns or causes cascading effects that indirectly impact replication.

• Complementation studies: Provide A18 function in trans (e.g., from a separate expression vector) to determine if defects can be rescued, indicating direct rather than indirect effects.

• Domain-specific mutations: Create targeted mutations in different functional domains of A18 to separate various activities and determine which are directly responsible for observed phenotypes.

• Genetic interaction mapping: Similar to studies of poxviral DNA polymerase and ribonucleotide reductase interactions , examine how A18 mutations interact with mutations in other viral genes to create an interaction network distinguishing direct from indirect effects.

• Immediate-early effects: Analyze the earliest detectable changes following A18 inhibition or mutation to identify direct targets versus downstream consequences.

• Physical interaction verification: Confirm direct protein-protein or protein-nucleic acid interactions using techniques such as crosslinking, chromatin immunoprecipitation, or proximity labeling.

These approaches can help researchers establish causality and mechanism in the complex regulatory network involving YMTV A18 protein.