Biology subjects

Shaw, A. B.

Publications and source records attributed to Shaw, A. B..

4 recordsLinked to original sources

bioRxiv2025

The orthobunyavirus Gc glycoprotein head and stalk drives an infectious virion assembly pathway that is specific for the insect host

The Orthobunyavirus genus of arthropod-borne segmented RNA viruses comprises several important pathogens including the human-infecting Oropouche virus and animal-infecting Schmallenberg virus (SBV). The prototypical Bunyamwera orthobunyavirus (BUNV) possesses envelope-embedded glycoprotein spikes, with Gc head and stalk domains forming distinctive tripods covering the envelope-proximal Gn. Spike ectodomains mediate virus entry, while endodomains interact with nucleoprotein (NP) enwrapped genome segments to mediate virion assembly. Interestingly, BUNV Gc head/stalk domains are redundant for virus growth in mammalian cells, consistent with isolations of SBV from animals bearing head/stalk deletions. However, orthobunyavirus isolations from arthropods in nature appear to maintain these domains strictly. To investigate this discrepancy, we compared the multiplication characteristics of wildtype BUNV (BUNV-WT) with a Gc head/stalk deleted BUNV (BUNV-{Delta}7). In mammalian cells BUNV-WT and BUNV-{Delta}7 grew to equivalent titres, whereas BUNV-{Delta}7 titres from insect cells were 1000-fold lower than BUNV-WT and strikingly produced no virions following blood meal infection of Aedes mosquitoes. To understand this insect-specific restriction in virion production, we showed the intracellular abundance of BUNV-WT and BUNV-{Delta}7 Gc and NP components were equivalent, suggesting {Delta}7-Gc was assembly-deficient. To explore this, we investigated Gc and {Delta}7-Gc interactions during BUNV-WT and BUNV-{Delta}7 infections of both insect and mammalian cells by co-immunoprecipitation and multiplex mass spectrometry, revealing {Delta}7-Gc exhibited markedly reduced NP interactions in insect cells. We propose Gc recruits genome segments during virion assembly and that the complete Gc head/stalk assembly is maintained in nature due to its essential role in the insect host. IMPORTANCEOrthobunyaviruses are arthropod-borne viruses that cause severe disease in humans and animals, including congenital malformations and abortions. Orthobunyavirus Gn and Gc glycoproteins form spikes, which mediate entry and genome recruitment during assembly. In nature, OBVs bearing large deletions within Gc head/stalk domains have been isolated from animals, yet the head/stalk domains appear to be strictly maintained within insects. To investigate this discrepancy, we compared the multiplication of Bunyamwera orthobunyavirus (BUNV-WT) with head/stalk-deleted variant (BUNV-{Delta}7). In mammalian cells both viruses reached similar titres, but strikingly BUNV-{Delta}7 failed to produce virions in both insect cells and Aedes mosquitoes. We showed this was because BUNV-{Delta}7 failed to assemble new virions, revealing its head/stalk-deleted Gc was deficient in interactions with genome components. We propose that Gc drives species-specific interactions with genome segments during virion assembly, explaining why Gc head/stalk domains are conserved in nature due to their essential role in the insect host.

microbiology↗

bioRxiv2023

Lymphocytic choriomeningitis arenavirus utilises tunnelling nanotube-like intercellular connections for cell-to-cell spread

The Arenaviridae family within the Bunyavirales order of segmented RNA viruses contains over 50 species grouped into four genera, Antennavirus, Hartmanivirus, Mammarenavirus and Reptarenavirus. Several mammarenaviruses are associated with fatal hemorrhagic fevers, including Lassa, Lujo and Junin viruses. The mammarenavirus member lymphocytic choriomeningitis virus (LCMV) is largely non-pathogenic to humans and represents a tractable model system for studying arenavirus molecular and cellular biology. During infection of cells in culture, a high proportion of LCMV spread is between directly neighbouring cells. Consistent with this observation LCMV-infected cells extrude multiple tunnelling nanotube (TNT)-like structures forming intercellular connections that could provide a route of cell-to-cell spread. To investigate this, we used recombinant LCMV with engineered epitope tags in glycoprotein spike (GP-1) and matrix (Z) proteins, alongside nucleoprotein (NP) antisera, to reveal that all three major structural proteins co-localised within TNT-like connections. Furthermore, utilising fluorescent in situ hybridisation (FISH) we showed NP also co-localised with LCMV genomic sense RNA. Taken together, these observations suggested LCMV virions pass between cells through intercellular connections to infect new cells. Consistent with this, addition of a potent LCMV neutralising antibody to supernatants during infection failed to block LCMV spread through cultures, revealing that cell-to-cell connectivity plays a major role in LCMV transmission. This is the first report of cell-cell infection via TNT-like connections for any species of the 14 families within the Bunyavirales order. This study furthers our understanding of how arenaviruses manipulate the host to establish infection, which may aid in the development of effective anti-viral therapeutics. IMPORTANCEArenaviruses include some of the most serious human pathogens in existence, although no clinically approved vaccines or therapies are currently available to prevent their associated disease. As with most pathogens, transmission of arenaviruses from one cell to another is a critical aspect of infection and resulting pathogenicity. Here, we showed that model arenavirus lymphocytic choriomeningitis virus (LCMV) can spread between cells without exposure to the extracellular space. We visualized the three major LCMV structural proteins, namely nucleoprotein, glycoprotein spike and matrix co-localized along with genomic RNA within tubular structures connecting adjacent cells. The use of a potent neutralizing antibody to block the extracellular route of LCMV transmission reduced spread within cultured cells to approximately half that of untreated cultures. Taken together, these results suggest intercellular connections represent important conduits for arenavirus spread. This information will aid in the development of antiviral strategies that prevent both intra- and extracellular transmission routes.

microbiology↗

bioRxiv2023

Lymphocytic choriomeningitis arenavirus requires cellular COPI and AP-4 complexes for efficient replication and virion production.

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromised populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within the Arenaviridae family. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatamer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harbouring a FLAG tagged GP-1 envelope spike (rLCMV-GP1-FLAG) we showed infection resulted in marked co-localization of COPI and AP-4 component with both LCMV nucleoprotein (NP) and GP-1. Time-of-addition studies using brefeldin A (BFA), an ARF-I inhibitor that prevents formation of both COPI and AP-4 complexes, suggested these cellular components were involved in late stages of the LCMV multiplication cycle. Consistent with this finding, BFA treatment at similar late time-points resulted in a marked redistribution of NP and GP-1, and subsequent loss of COPI/AP-4 co-localization. Finally, titration of released virus within supernatant of BFA-treated cells revealed a 10-fold decrease in viral titres, greater than the 2-fold BFA-mediated reduction in NP expression. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors that are required for efficient LCMV assembly and egress. ImportanceArenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member LCMV being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. Here, we assessed the impact of siRNA knockdown of host cell trafficking genes on LCMV multiplication. We reveal the requirement of host cellular COPI and AP-4 complexes for efficient LCMV multiplication, acting late in the replication cycle, at the stages of egress and assembly. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal novel cellular trafficking pathways required during infection. Moreover, this study may lead to the discovery of novel therapeutic targets for arenaviruses to prevent serious human disease.

microbiology↗

bioRxiv2023

Cellular endosomal potassium ion flux regulates arenavirus uncoating during virus entry

Lymphocytic choriomeningitis virus (LCMV) is a model arenavirus that causes fatalities within immunocompromised populations. To enter cells, the LCMV envelope fuses with endosomal membranes, for which two requirements are low pH and interaction between LCMV GP spike and receptor CD164. LCMV subsequently uncoats, where genome-associated NP separates from Z matrix. To further examine LCMV entry, an siRNA screen identified K+ channels as important for LCMV infection, and pharmacological inhibition confirmed K+ involvement during entry. We tracked incoming virions along their entry pathway under physiological conditions, where uncoating was signified by separation of NP and Z. In contrast, K+ channel blockade, prevented uncoating, trapping virions within Rab7 and CD164-positive endosomes, identifying K+ as a third LCMV entry requirement. K+ did not increase GP/CD164 binding, thus we suggest K+ mediates uncoating by modulating NP/Z interactions within the virion interior. These results suggest repurposing licensed K+ channel inhibitors represents a potential anti-arenaviral strategy.

cell biology↗