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Lassa virus spike complex for structure and receptor recognition: A step toward vaccine designing

Lassa haemorrhagic fever (LF) is a deadly disease caused by the Lassa virus (LASV). LASV is indigenous to West Africa, although it has recently expanded to other areas. Although zoonotic transmission from rodents is the most common route of infection, human-to-human transfer has resulted in a significant number of cases, raising the possibility of massive outbreaks. Ribavirin is the only medication for Lassa haemorrhagic fever that has been approved by the US Food and Drug Administration so far, however it has side effects and its efficacy is unknown. As a result, filling the gaps in our knowledge about this fatal disease is critical in guiding the development of better medicines.

Before reaching its mature state, all arenaviruses' spike complexes go through a lot of cellular processing. The homotrimeric complex protomers are initially produced as glycoprotein precursor proteins (GPCs), then cleaved twice by a signal peptidase (SPase) and a subtilisin kexin isozyme-1/site-1 (SKI-1) protease. The stable signal peptide (SSP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2) are the three functional subunits that result from this processing (Fig. 1a). The receptor-binding domain of GP1 is responsible for cell receptor attachment.

The structural role of arenaviruses' abnormally lengthy stable signal peptide (SSP) has remained a mystery. Because  SSP is so conserved in arenaviruses, it's very likely that they all have the same SSP arrangement, as the team discovered. these finding were publish in "Nature".

The second hydrophobic region of SSP, according to team findings, is not connected with the spike's central transmembrane region and is found in the cytoplasm or inside the virion. The arrangement of the SSP suggests that there are roughly 25 SSP residues inside the cell, in addition to the 38 GP2 residues that constitute a cytoplasmic zinc-binding domain. As a result, arenavirus spike complexes have a large protein mass in the intraviral–inner-cellular area.

The separation of SSP's C terminus and GP1's N terminus across the membrane implies a topological rearrangement event that occurs following SPase cleavage. It's worth noting that topological variations in signal peptides have been reported before, which could explain why identifying the topology of the SSP experimentally is so challenging. SSP's association mode with the trimeric spike suggests that it aids in maintaining the native structure's stability by keeping the transmembrane helices of GP2 in place.

According to authors "We believe that structural changes to the spike occur during priming, and that these changes may involve changes in the way SSP interacts with the spike. This could help explain how SSP residues like Lys33 affect pH-induced spike triggering.

Other factors may influence the binding strength of matriglycan by the LASV spike complex, in addition to the numerous polar contacts and the prolonged buried surface area that drive the binding of matriglycan by the LASV spike complex. Multiple degenerate binding states result from the interaction of the LASV spike complex, which has three-fold symmetry and contains a linear asymmetric form of matriglycan. 

Despite the fact that these states are totally identical and indistinguishable, their existence reduces the entropic cost of binding significantly. Additional states could be permitted utilizing similar geometry in addition to the observed conformation in our structure.

Two or even three different matriglycan chains might theoretically attach to a single trimer at the same time, resulting in numerous degenerate states. Simultaneous binding of many chains may be critical for establishing the virus's initial interaction with the host cell, and if the matriglycan chains are fixed to the same surface, avidity should help.

It's also possible that the spike complex will bind in the middle of a long matriglycan chain. A geometry for such a bond has been presented. This hypothetical connection could allow virions to slide along long matriglycan chains, similar to how proteins slide along other biological polymers.

The discovery that the RRLL–SKI-I recognition motif has essential structural roles in maintaining the spike and generating the matriglycan-binding site explains why SKI-function I's in cleaving the GPC has remained evolutionary conserved. Obtaining a furin cleavage site boosts the pathogenicity of other viruses that use class-I spike complexes, such as SARS-CoV-2, by allowing NRP1 and NRP2 to recognize the newly generated C′ and operate as attachment factors. The newly generated C′ must remain exposed in the mature spike to allow binding by the NRPs, which is true for SARS-CoV-2 but not for LASV. 

When a furin site is artificially added to the spike complex of LASV, it results in a cleaved trimer that lacks the stabilising swapping mechanism and has a defective receptor binding site, as the team discovered. During biogenesis, viruses are known to 'steal' molecules from their host cells. The 'pocket factor,' a cellular-derived fatty acid that binds and stabilizes rhinovirus coat proteins, is a well-known example. 

The spike complex of SARS-CoV-2, for example, needs linoleic acid from its host cell to maintain a 'closed' shape. The LASV spike complex hooks on to what seems to be a free matriglycan polymer after maturation. There are two crucial prerequisites for this to happen: (1) Matriglycan must be accessible, and (2) the spike complexes must be able to bind it. SKI-I is found in the Golgi, where it processes the LASV GPC at the endoplasmic reticulum–cis-Golgi junction.

LARGE1 is an enzyme that makes matriglycan and is also found in the Golgi. LARGE1 does, however, produce matriglycan on -DG rather than as a free polymer in solution. Incubating the enzyme 1,4-glucuronyltransferase (B4GAT1) with Xyl and UDP-GlcA in vitro results in a GlcA–1-4-Xyl, which LARGE1 recognizes and elongates. As a result, B4GAT1 in the Golgi might theoretically produce free substrates that LARGE1 could then elongate to form free matriglycan polymers.

The research team propose that the LASV spike reaches a Golgi compartment where free matriglycan polymers are available after SKI-I-dependent maturation at the endoplasmic reticulum–cis-Golgi, and that this is where loading happens. The attached matriglycan acts as a competitive inhibitor of matriglycan binding on cells. Using free matriglycan to inhibit cell attachment for a set period of time minimises the likelihood of newly produced -DG-tropic arenaviruses sticking back to their host cells, allowing for easier viral escape. 

Furthermore, because the LASV spike complex is primed in a low-pH endocytic environment after cell entry, the presence of free matriglycan in the trans-Golgi network—which is similarly characterized by low pH—could assist stabilize the spike and prevent it from priming prematurely.

Introducing various modifications to viral spike proteins, such as truncations, point mutations, and tweaks, has been a key method for getting structural information. As shown in the instance of the LASV spike, such alterations may accidentally modify important structural aspects such as the development of the matriglycan-binding site. The use of single-particle cryo-EM may be able to avoid or at least lessen the requirement for such modifications. The discovery of the matriglycan-binding site and the shape of a persistent LASV spike may aid in the development of better therapeutics for this high-priority human disease.

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