Biology subjects

Barr, J. N.

Publications and source records attributed to Barr, J. N..

5 recordsLinked to original sources

bioRxiv2024

Single cell variation and rapid emergence of phenotypic heterogeneity in cell lines- a cautionary tale for devotees of CRISPR-Cas9.

The use and publication of research utilising CRISPR/cas9 in gene editing and knock outs (KOs) within cell lines is now widespread and has proved extremely powerful in the interrogation of gene function. However, the potential of experimental artefacts resulting from the need to generate single cell clones post gene-manipulation has been somewhat overlooked. In this study, we show that the commonly used pulmonary cell line A549 displays significant heterogeneity in terms of their gene expression, and that individual cells from a population exhibit dramatically different susceptibility to viral infection from a range of viruses including respiratory syncytial virus (RSV), influenza A virus (IAV), Hazara virus (HAZV), lymphocytic choriomeningitis virus (LCMV) and Bunyamwera virus (BUNV). Moreover, we demonstrate the rapid re-emergence of phenotypic heterogeneity even after cloning. These results demonstrate the need for caution in interpreting results from CRISPR screens and CRISPR-KO validation studies, especially in the study of viral infection.

microbiology↗

bioRxiv2023

The cryoEM structure of the Hendra henipavirus nucleoprotein reveals insights into paramyxoviral nucleocapsid architectures

We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 [A] resolution, derived from single particle analysis of homotetradecameric RNA-bound N protein rings exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent N and N+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional interaction between N+1 and N-1 protomers. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.

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↗