Tick Viruses Have a Secret Weapon: A Baffling Double Loop RNA Structure!

You know, the world of viruses is just wild. They’re constantly finding new ways to mess with us, and honestly, you’ve got to admire their ingenuity, even if they make us sick. We’re talking about the Flaviviridae family here – the gang that includes notorious characters like Zika, Dengue, Yellow Fever, and yes, those nasty tick-borne ones like Powassan Virus (POWV).

These viruses are clever. When they infect a cell, they don’t just make more virus particles; they also churn out these little side projects called subgenomic flaviviral RNAs, or sfRNAs. Think of them like viral propaganda or sabotage tools. How do they make them? Well, our cells have this cleanup crew, enzymes called exoribonucleases (like the main one, Xrn1), that chew up RNA from one end to the other. But these viruses put up roadblocks!

These roadblocks are special structured RNA elements in the virus’s genetic material, specifically in the 3′ untranslated region (UTR). Scientists call them exoribonuclease resistant RNAs, or xrRNAs. Their whole job is to stop our cellular cleanup crew (Xrn1) dead in its tracks, preventing the full degradation of the viral RNA and thus creating those sfRNAs. What’s really fascinating is that these xrRNAs work *by themselves*, purely based on their 3D shape, without needing any viral or host proteins to hold their hand.

The Known Players: Mosquito-Borne xrRNAs

For a while now, we’ve had a decent picture of how the xrRNAs from mosquito-borne flaviviruses (like Zika) work. Their structures, when solved, show this really cool, conserved fold. The defining feature? A unique ring-like motif that literally encircles the 5′ end of the xrRNA. Imagine a tiny molecular bracelet. This ring acts like a brace, jamming up against the surface of our Xrn1 enzyme and physically stopping it from moving forward and unwinding the RNA.

This ring structure is stabilized by all sorts of intricate molecular origami – things like pseudoknots (where one part of the RNA folds back and pairs with another distant part), base triples, and other non-standard base pairings. It’s a complex, beautiful little machine designed purely to be a pain in Xrn1’s neck.

The Tick-Borne Mystery

But here’s the twist: the xrRNAs found in tick-borne flaviviruses (TBFV) and those with no known vector (NKVFV) seemed… different. Bioinformatic analysis hinted at variations in their size and secondary structure (how the RNA folds into stems and loops). They didn’t quite fit the mold of the mosquito-borne ones (which are classified as Class 1, with subclasses 1a and 1b). The tick-borne ones fell into Class 2, and their 3D structure? Totally unknown. This was a big gap in understanding how these specific, often encephalitis-causing, viruses pulled off their trick.

So, the big question was: Do tick-borne flaviviruses even *have* functional xrRNAs? And if so, what do they look like in 3D? Do they use the same ring strategy, or something completely different?

Cracking the Code: Enter Powassan Virus

To figure this out, scientists turned their attention to Powassan Virus (POWV). POWV is a serious tick-borne pathogen in North America and Asia, and frankly, we need to understand its tricks better, especially since there aren’t specific treatments for it. First, they had to confirm that POWV actually produces sfRNAs during infection, which they did using standard lab techniques like Northern blots. Yep, POWV makes sfRNAs, just like its mosquito cousins.

Then, they verified that these sfRNAs were indeed the result of xrRNAs blocking Xrn1. They did this with an in vitro (in a test tube) experiment, showing that purified Xrn1 enzyme stopped at specific points on the POWV RNA, leaving behind the expected sfRNA fragments. This confirmed that POWV has authentic xrRNAs that function on their own, without needing help from other molecules.

Mapping the Halt Site: A Clue to the Difference

With Class 1 xrRNAs, Xrn1 stops just *before* the ring structure, in an unpaired region. The ring is pre-formed and essentially impenetrable. But with the proposed structure of Class 2 xrRNAs, there was a hint that a key structural element called Pseudoknot 1 (PK1) was much longer than in Class 1. What’s more, previous work suggested the stop site for Xrn1 on Class 2 xrRNAs might actually be *within* this longer PK1.

Mapping the exact stop sites on the POWV xrRNAs confirmed this suspicion. Unlike the precise, single stop site seen in Class 1, Xrn1 stopped at *multiple* positions spread out within the predicted PK1 region of the POWV xrRNAs. This is a big deal because Xrn1 can only chew up single-stranded RNA. To reach a stop site *inside* a base-paired pseudoknot, the enzyme must be unwinding the RNA structure as it goes! This immediately suggested that the interaction between Xrn1 and the Class 2 xrRNA is different from Class 1.

Seeing the Structure: Cryo-EM Reveals the Double Loop

Getting a 3D structure of RNA can be tricky, especially for Class 2 xrRNAs which hadn’t yielded to traditional methods like crystallography. So, the researchers turned to cryo-electron microscopy (cryo-EM), a powerful technique that can image biological molecules at near-atomic resolution. To make the relatively small xrRNA easier to see with cryo-EM, they used a clever trick: they attached the xrRNA to a larger, known RNA structure (a ribozyme scaffold). This is like putting a tiny object on a bigger handle so you can grab and image it better. Crucially, they showed this attachment didn’t mess up the xrRNA’s function.

The cryo-EM maps, even at mid-resolution, were enough to see the overall shape of the POWV xrRNA. And wow, did it reveal something unique! The core of the structure was similar to one of the Class 1 subclasses (Class 1b), but the famous ring-like feature? It was *much* more extensive than anything seen before. Instead of just wrapping around the 5′ end once, the POWV xrRNA structure showed the RNA looping around *twice*. They dubbed this a double loop ring.

Putting it Together: Structure, Bioinformatics, and Function

To really understand this double loop and confirm it wasn’t just a weird artifact of the POWV xrRNA they studied, they combined the cryo-EM data with other evidence:

• Bioinformatics: They used the structural insights to refine searches for other Class 2 xrRNAs. They found more examples in various tick-borne and no-known-vector flaviviruses. A detailed covariation analysis (looking for pairs of nucleotides that change together across different virus sequences, indicating they’re base-paired or interacting) strongly supported the proposed secondary structure, including the extended PK1 and the elements forming the double loop. This showed the POWV structure is representative of the whole Class 2.
• Chemical Probing: Using chemical tools that react differently with paired vs. unpaired or flexible RNA regions, they confirmed the secondary structure model and found some dynamic regions, particularly near the 5′ end of PK1 – exactly where Xrn1 seems to stop!
• Functional Tests: They made mutations to disrupt key predicted interactions, like the extended PK1 and conserved nucleotides (especially three specific ‘A’ bases that are found in *all* Class 2 xrRNAs). Disrupting these elements caused the xrRNA to lose its ability to block Xrn1, proving their importance for function.

A New Model for Resistance

So, what does this double loop mean for how Class 2 xrRNAs block Xrn1? The old model for Class 1 was a rigid brace. The new model for Class 2, based on the double loop structure and the halt site location *within* PK1, is different. Imagine Xrn1 chugging along the RNA. When it hits the Class 2 xrRNA, it starts trying to unwind the extended PK1. As it unwinds, the RNA that forms the double loop gets pushed and potentially remodeled between the enzyme and the core of the xrRNA structure.

This forced remodeling and the resulting steric clash (basically, molecules bumping into each other where they shouldn’t) is likely what jams the enzyme. The fact that Xrn1 stops at multiple points within PK1 could be explained by this process – maybe the enzyme struggles and “stutters” as it tries to unwind and push through the double loop. It’s a more dynamic interaction compared to the seemingly rigid block of Class 1.

Why Does This Matter?

Understanding the 3D structure of Class 2 xrRNAs fills a crucial gap. It shows that while all flavivirus xrRNAs use a similar overall strategy – a structured RNA ring blocking an enzyme – they’ve evolved different ways to achieve it. The Class 1 uses a single, tight loop, while Class 2 uses an extended PK1 to create a remarkable double loop. This highlights the incredible structural flexibility of RNA and how evolution can arrive at similar functional outcomes via different structural paths.

Knowing these structures and mechanisms is vital. These xrRNAs and the sfRNAs they produce are linked to various ways these viruses cause disease and evade our immune system. By understanding the fundamental molecular machines the virus uses, we might be able to design new ways to disrupt them. Perhaps a drug could be developed that specifically targets the unique double loop of tick-borne flavivirus xrRNAs, leaving our own cellular RNAs and mechanisms untouched. That’s the hope, anyway!

It’s a complex puzzle, but uncovering structures like this double loop gives us powerful new pieces to work with in the fight against these important global health threats.

Source: Springer