Recombinant His1 virus Putative transmembrane protein ORF17 (ORF17)

What is ORF17 and what roles does it play across different viral families?

ORF17 serves distinct functions depending on the viral species. In Kaposi's sarcoma-associated herpesvirus (KSHV), ORF17 encodes a viral protease precursor (ORF17-prePR) that contributes significantly to capsid formation and maturation. This protease precursor undergoes functional cleavage into a protease (ORF17-PR) and an assembly region (ORF17-pAP/-AP) . The protease function is essential for appropriate capsid maturation, with studies showing that wild-type KSHV produces mature capsids, whereas ORF17-deficient and protease-dead KSHV produces only B-capsids (closed bodies possessing a circular inner structure) .

In contrast, Varicella-zoster virus (VZV) ORF17 functions differently, showing homology to herpes simplex virus (HSV) UL41, which encodes the viral host shutoff protein (vhs). The VZV ORF17 protein induces RNA cleavage, although to a substantially lesser extent than HSV-1 vhs . VZV ORF17 is predicted to encode a 455-amino-acid protein with 39% amino acid identity to HSV-1 vhs, sharing four highly conserved domains .

How are ORF17 proteins structurally characterized?

ORF17 proteins exhibit important structural differences across viral species that correlate with their functional diversity. VZV ORF17 and HSV-1 vhs have distinctive deletion patterns: HSV-1 vhs has a deletion corresponding to VZV ORF17 amino acids 170 to 189, while VZV ORF17 protein has a deletion corresponding to HSV-1 vhs amino acids 306 to 358 .

The functional domains also differ significantly. HSV-1 vhs contains a domain that binds to HSV VP16 and regulates vhs activity, but VZV ORF17 protein lacks this binding domain despite the virus encoding a VP16 homolog (the VZV ORF10 protein) . Furthermore, the poly(A) binding domain LGYAYIN in HSV-1 vhs is only partially conserved (MGYAYVE) in VZV ORF17, which may explain the reduced RNA cleavage activity observed in VZV ORF17 .

In KSHV, the restriction or release site (R-site) in ORF17 is particularly critical, as mutations at this site prevent the functional cleavage of ORF17-prePR into separate protease and assembly components, significantly impairing viral production .

How are ORF17 deletion mutants constructed for functional studies?

Researchers have employed several sophisticated genetic engineering approaches to generate ORF17 mutants:

For KSHV, researchers have created:

• Complete ORF17-deficient viruses

• ORF17 protease-dead viruses with specific mutations affecting enzymatic activity

• ORF17 R-mut variants with a point-mutation at the restriction/release site that prevents functional cleavage

For more complex studies examining the interplay between ORF17 and other viral proteins, double knockout viruses (ORF7&17-DKO) have been constructed using a two-step markerless Red recombination system. This process typically begins with single knockout constructs (e.g., ORF7-fsKO-BAC16) followed by insertion of the ORF17 knockout frameshift mutation, with confirmation by Sanger sequencing .

The specific construction of ORF7&17-DKO-BAC16 illustrates this approach: "ORF7&17-DKO-BAC16 was generated from ORF7-fsKO-BAC16 using a two-step markerless Red recombination system... The mutagenesis of these BAC clones were performed according to a previously described protocol, using specific mutagenesis primers" .

What assays are used to evaluate ORF17 function in viral replication?

Researchers employ multiple complementary approaches to assess ORF17 function:

• Viral production quantification: Measuring viral DNA in culture supernatants using quantitative PCR (qPCR) to determine how ORF17 mutations affect viral yield .

• Infectivity assays: Adding culture medium containing virus to uninfected cells (e.g., HEK293T) and counting GFP-positive cells by flow cytometry to evaluate infectious virion production .

• Transcriptional analysis: Employing real-time reverse-transcription PCR (qRT-PCR) to evaluate expression levels of immediate early (IE), early (E), and late (L) genes in wild-type versus ORF17-deficient viruses .

• Ultrastructural analysis: Using transmission electron microscopy (TEM) to directly observe capsid morphology differences between wild-type and mutant viruses, which provides visual evidence of ORF17's role in capsid maturation .

• Temperature-dependent growth assays: Comparing viral growth at different temperatures (33°C vs. 37°C) to identify temperature-sensitive functions of ORF17, as demonstrated in VZV studies .

• Protein expression confirmation: Using Western blotting to verify expression of wild-type and mutant ORF17 proteins in infected or transfected cells .

• In vivo models: Employing animal models such as cotton rats to study aspects like latent infection establishment in the presence or absence of functional ORF17 .

What is the role of ORF17 in capsid assembly and maturation?

ORF17 plays a critical role in herpesvirus capsid maturation through its protease activity. In KSHV, ORF17 functions as a viral protease that digests scaffold proteins, resulting in scaffold shell disruption - a key step in capsid maturation. Studies with ORF17-deficient KSHV demonstrated that capsid formation becomes arrested between the procapsid and B-capsid stages .

The importance of the protease function specifically was confirmed through the creation of protease-dead KSHV variants, which showed decreased viral production without affecting DNA replication . Structurally, wild-type KSHV produces mature capsids, whereas ORF17-deficient and protease-dead KSHV produce only B-capsids, which are closed bodies possessing a circular inner structure .

Complementation assays further validated ORF17's essential role: "Virus production in ORF7&17-DKO-iSLK cells recovered significantly when ORF7-2xS (ORF7-2S) and 3xFLAG-ORF17 (3F-ORF17) were exogenously co-expressed... The infectivity of the virions produced from ORF7&17-DKO-iSLK cells were recovered by exogenous ORF7-2xS (ORF7-2S) and 3xFLAG-ORF17 (3F-ORF17) co-expression" .

How does ORF17 contribute to RNA processing in viral systems?

The RNA processing function of ORF17 varies significantly between viral species. In VZV, ORF17 protein induces RNA cleavage similar to but less effectively than its HSV-1 counterpart (vhs). Experimental data shows that while HSV-1 vhs abolishes expression from a β-galactosidase reporter plasmid, VZV ORF17 does not inhibit expression from the same reporter system .

This functional difference appears to correlate with structural variations. The HSV-1 vhs contains a conserved domain (LGYAYIN) postulated to bind to poly(A) sequences, but this sequence is only partially conserved (MGYAYVE) in VZV ORF17 . These structural differences likely account for the observed functional variations in RNA processing activity.

Unlike HSV vhs, which is located in virions, VZV ORF17 protein was not detectable in virions, suggesting different mechanisms of action in the viral replication cycle .

How does temperature affect ORF17 function and viral replication?

Temperature dependency represents one of the most fascinating aspects of ORF17 function, particularly in VZV. Research demonstrates that while ORF17 VZV mutants grew to peak titers similar to the parental virus at 33°C, they showed dramatically reduced growth (20- to 35-fold lower titers) compared to the parental virus at 37°C .

This temperature sensitivity extends beyond simple growth kinetics to affect protein localization patterns. Studies revealed that ORF62 protein (another viral protein) exhibited a significantly different distribution pattern in the nuclei and cytoplasm of cells infected with an ORF17 deletion mutant at 37°C compared to cells at 33°C .

These findings suggest that ORF17 may have evolved to facilitate virus growth in specific tissues with particular temperature conditions, which has important implications for understanding viral tropism and pathogenesis in vivo .

What is the sequential relationship between ORF17 and other viral proteins in morphogenesis?

Determining the temporal sequence of events in viral assembly represents a significant research challenge. In KSHV, researchers have employed elegant genetic approaches to establish the order of actions between ORF7-mediated genome cleavage and ORF17-mediated internal scaffold disruption during capsid assembly.

The key experimental design involved comparing phenotypes between single and double knockout viruses: "If ORF7 acts earlier than ORF17 in capsid formation, then the ORF7&17-DKO KSHV should have the same phenotype as ORF7-KO KSHV. On the other hand, if ORF17 acts earlier than ORF7, then the ORF7&17-DKO KSHV should present the same phenotype as ORF17-KO KSHV" .

Results demonstrated that "ORF7&17-DKO KSHV showed the same properties as ORF7-KO KSHV in viral genome replication and infectious virus production. These phenotypes are fully consistent with those of ORF17-KO KSHV" . This evidence established that ORF17 acts earlier than ORF7 in the capsid formation process, providing critical insights into the sequential events of herpesvirus morphogenesis.

How do ORF17 mutations impact in vivo viral pathogenesis?

Research on the in vivo consequences of ORF17 mutations has yielded intriguing results that sometimes contradict in vitro findings. While ORF17 appears essential for optimal viral replication in cell culture systems, particularly at physiological temperatures, its role in establishing latent infection may be more nuanced.

In VZV research, "inoculation of cotton rats with the ORF17 deletion mutant resulted in a level of latent infection similar to that produced by inoculation with the parental virus" . This surprising finding suggests that despite growth defects in cell culture at 37°C, ORF17-deficient viruses may still establish latency effectively in vivo.

This apparent contradiction highlights the complexity of viral-host interactions in living organisms compared to cell culture systems and underscores the importance of animal models in comprehensively understanding viral protein functions.

What structural studies would advance understanding of ORF17 function?

Advanced structural biology approaches could significantly enhance our understanding of ORF17 function. High-resolution structural determination of ORF17 proteins, particularly focusing on the protease domain in KSHV ORF17 and the RNA-binding regions in VZV ORF17, would provide crucial insights into their mechanism of action.

Comparative structural studies between temperature-sensitive VZV ORF17 at both permissive (33°C) and non-permissive (37°C) temperatures could potentially reveal conformational changes that explain the observed temperature dependency .

Additionally, structural characterization of ORF17 in complex with its substrate proteins during capsid assembly would illuminate the precise mechanism of scaffold protein processing and capsid maturation.

What are the key unanswered questions about ORF17 evolution?

The functional diversity of ORF17 proteins across different viral families raises fascinating evolutionary questions. Understanding how these proteins evolved from a common ancestor to perform distinct functions (protease activity in KSHV versus RNA processing in VZV) would provide insights into viral adaptation.

Comparative genomic analyses across the herpesvirus family could trace the evolutionary trajectory of ORF17, identifying key mutation events that led to functional divergence. This evolutionary perspective might reveal how selective pressures shaped ORF17 function in different viral ecological niches.

The temperature sensitivity of VZV ORF17 represents a particularly interesting evolutionary adaptation that warrants further investigation in the context of viral host range and tissue tropism.