Biology subjects

Crothers, J. W.

Publications and source records attributed to Crothers, J. W..

4 recordsLinked to original sources

bioRxiv2025

Rab11B is required for binding and entry of recent H3N2, but not H1N1, influenza A isolates

As an obligate intracellular parasite, influenza A virus (IAV) depends on host proteins to complete several important functions, including trafficking viral proteins throughout the cell. Previous work has established a critical role for the cellular vesicular trafficking protein, Rab11A, in transporting the viral genome segments to the site of budding at the plasma membrane. While the role of Rab11A in IAV assembly is relatively well understood, very little is known about the function of a closely related isoform (Rab11B) during influenza virus infection. We have shown that both Rab11A and Rab11B are required for successful IAV infection by current H1N1 or H3N2 isolates. Cells in which either Rab11A or Rab11B were depleted failed to efficiently produce virus, with significant reductions in infectious titer. Surprisingly, our data revealed that recent (2022) H3N2, but not H1N1, isolates failed to efficiently produce viral proteins in single-cycle infections when Rab11B (but not Rab11A) was depleted. Flow cytometry analysis suggests that the defect in protein production is driven by a reduction in the total number of infected cells, rather than a decrease in viral protein production at the single cell level. Using reverse genetics and 7+1 reassortant viruses we mapped this Rab11B-dependent early defect in recent H3N2 isolates to the HA gene. RT-qPCR analysis of H3N2 virions bound to the cell surface showed a [~]50% decrease in virus binding the surface of cells depleted of Rab11B, but not Rab11A. This data suggests a novel role for Rab11B during viral entry, likely at the stage of viral binding, that is specific to H3N2 isolates.

microbiology↗

bioRxiv2024

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

microbiology↗

bioRxiv2020

RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWAB USING QIAGEN RNEASY KITS OR DIRECTLY VIA OMISSION OF AN RNA EXTRACTION STEP

The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.

microbiology↗

bioRxiv2019

Dynamic colonization of microbes and their functions after fecal microbiota transplantation for inflammatory bowel disease

For fecal microbiota transplantation (FMT) to be successful in complex immune diseases like inflammatory bowel disease (IBD), it is assumed that therapeutic microbes and their beneficial functions and immune interactions must colonize the recipient and persist in sufficient quantity and for a long enough period of time to result in a clinical benefit. But few studies have comprehensively profiled the colonization and persistence of transferred microbes along with the transfer of their microbial and immune functions. Using 16S, metagenomic, and immunoglobulin A (IgA) sequencing, we analyzed hundreds of longitudinal microbiome samples from a randomized controlled trial of 12 patients with ulcerative colitis who received fecal transplant or placebo for 12 weeks. We uncovered a range of competitive dynamics among donor and patient strains, showing that persistence of transferred microbes is far from static. Indeed, one patient experienced dramatic loss of donor bacteria 10 weeks into the trial, coinciding with a bloom of pathogenic bacteria and worsening clinical symptoms. We similarly evaluated transfer of microbial functions, including desired ones like butyrate production and unintended ones like antibiotic resistance. By profiling bacteria coated with IgA, we identified IgA-coated bacteria associated with inflammation, and we found that microbial interactions with the host immune system can be transferred across people. This transfer of immune function is likely critical for gut microbiome therapeutics for immune-related diseases. Our findings elucidate the colonization dynamics of gut microbes as well as their functions in the context of FMT to treat a complex disease--information that may provide a critical foundation for the development of more-targeted therapeutics.

microbiology↗