Biology subjects

Bair, C. R.

Publications and source records attributed to Bair, C. R..

5 recordsLinked to original sources

bioRxiv2026

E. coli RsmF activity depends on prior modification of 16S rRNA helix 44

Bacterial ribosomal RNA (rRNA) methylations are important for accurate translation. Four distinct methylations incorporated by RsmE, RsmF, and RsmH/ RsmI form a cluster of three modified 16S rRNA nucleotides (m3U1498, m5C1407, and m4Cm1402) surrounding the decoding center of the 30S subunit. Given their common substrate requirement of a late-stage intermediate 30S subunit, these enzymes likely act contemporaneously during subunit biogenesis, but whether there exists a required modification order is unknown. Here, using hypomethylated 30S subunits obtained from a collection of rsmH/I/E/F-deleted Escherichia coli strains, we identify RsmF activity to be highly dependent on prior modification of h44 both in vitro and in E. coli. RsmF activity on hypomethylated 30S subunits could be partially rescued by prior in vitro methylation using RsmE and RsmH, indicating that incorporation of these methyl groups directly shapes h44 for recognition by RsmF. RNA structure probing using SHAPE-MaP and molecular dynamics simulations reveal specific alterations in 16S rRNA structure and dynamics in the absence of the m4C1402 (RsmH) and m3U1498 (RsmE) modifications that likely restrict RsmF action. These studies thus uncover a previously unappreciated "order of operations" for 16S rRNA modification during ribosome biogenesis with important implications for studies on the collective functions of these modifications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/736617v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@f843cborg.highwire.dtl.DTLVardef@12acb13org.highwire.dtl.DTLVardef@7a8d9eorg.highwire.dtl.DTLVardef@1e7aa5f_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LI16S rRNA C1407 modification by RsmF depends on the prior action of RsmE and RsmH in E. coli C_LIO_LIm5C1402/ m3U1498 alter 16S rRNA nucleotide dynamics creating a 30S substrate suitable for RsmF C_LIO_LIAn order of operations exists for h44 modifying enzymes acting at the 30S subunit decoding center C_LI

biochemistry↗

bioRxiv2026

Comparative analysis of flavivirus sfRNA dynamics and secondary structure

The accumulation of subgenomic flavivirus RNAs (sfRNAs) modulates viral fitness and pathogenicity in culture and in vivo. These noncoding RNAs are produced by incomplete digestion of the flavivirus genome by the cellular 5-3 exoribonuclease (XRN1). Diverse flaviviruses have conserved RNA structural elements (RSEs) that map to their 3-untranslated region (3-UTR): Xrn-resistant RNA structures, dumbbell structures, and a 3-stem loop (3SL). Despite the importance of the 3-UTR RSEs for flavivirus replication, the structural dynamics of sfRNA during flavivirus infection are understudied. Here, we use digital droplet PCR to quantify sfRNA levels during infection for a panel of mosquito-borne flaviviruses (MbFV) including dengue virus serotypes 1 (DENV1), 2 (DENV2), and 4 (DENV4), and Zika virus (ZIKV). We then used SHAPE-MaP on XRN1-digested, in vitro-transcribed sfRNAs from each virus to determine their secondary structures compared to the corresponding sfRNAs obtained from flavivirus-infected A549 cells. Results seen in-cell and in vitro were largely similar; however, motifs within the dumbbell, the small hairpin (sHP) directly upstream of the 3-SL, and 3-SL regions showed significant differences in the extent of nucleotide reactivity. These differences were consistent among the four flaviviruses examined and may indicate regions of sfRNA that are shielded by interaction with proteins or other nucleic acids during infection. However, strong protection indicative of sustained interaction was not apparent. Our findings suggest that sfRNA interactions with viral and host factors within the cell are few, occur via base-paired regions, or are highly transient. ImportanceFlaviviruses are highly prevalent human pathogens. The flavivirus genome contains RNA structural elements (RSEs), including those encoded in the 3-UTR, that are necessary for viral replication. Subgenomic flavivirus RNAs (sfRNAs) are produced by incomplete digestion of flavivirus genomic RNA due to the cellular exoribonuclease XRN1 encountering 3-UTR RSEs that promote its stalling and disassociation. Viruses unable to produce sfRNAs are highly attenuated, underlining their biological importance. sfRNA secondary structure has been investigated previously but little information is available on sfRNA secondary structure dynamics in infected cells. By comparing SHAPE-MaP reactivities in vitro and in cells, we determined that previously inferred structures are likely maintained within infected cells. We also identified differences in the extent of SHAPE reactivity between in vitro and in-cell environments that were common to multiple mosquito-borne flaviviruses. These differences suggest that sfRNAs may engage in transient interactions within the cell that may be important for their function.

microbiology↗

bioRxiv2026

Conserved stem-loops of the SARS-CoV-2 5'-UTR activate OAS1

The innate immune system relies on pathogen recognition receptors, such as the 2,5-oligoadenylate synthetase (OAS) proteins, to detect pathogen-associated molecular patterns like viral double-stranded (ds)RNA. A specific splicing variant of OAS1 (OAS1-p46) has been implicated in initiating an immune response that leads to decreased disease severity during SARS-CoV-2 infection. OAS1-p46 has a C-terminal lipid modification motif that allows for anchoring of the protein to intracellular membranes and thus potential colocalization with immunogenic viral RNA regions such as the SARS-CoV-2 5-untranslated region (5-UTR). Here, we show that OAS proteins can detect the 5-structured elements (5-SE)-comprising the 5-UTR and three additional stem-loop structures-and activate the cellular RNase L pathway. Through systematic 3-end truncations of the 5-SE, we show that the smallest 5-SE fragment capable of potently activating OAS1 is the conserved and highly structured SL1-4b region, containing the first four stem-loop structures and their intervening linking sequences (SL1-4), as well as an unstructured region ("SL4b") to the 3-side of SL4. Analyses of OAS1 activation and RNA secondary structure probing using selective 2-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) of additional SL1-4b RNA variants suggests a model in which SL4 acts as the primary OAS1 interaction site, while the unstructured SL4b region and two other stem-loops (SL1 and SL3) are necessary for optimal presentation of this region for OAS1 activation. Our findings reveal a structurally complex viral RNA region that potently activates OAS1, underscoring the potential complexity of RNAs that can strongly activate this innate immune sensor.

biochemistry↗

bioRxiv2021

Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission

Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing. However, contrary to expectation, trends observed with sub-optimal exposure timing suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.

microbiology↗

bioRxiv2020

Human Adenovirus Type 4 Comprises Two Mayor Phylogroups With Distinct Phenotypes Relevant To Pathogenesis

Human adenovirus type 4 (HAdV-E4) is the only type (and serotype) classified within species Human mastadenovirus E that has been isolated from a human host to the present. Recent phylogenetic analysis of whole genome sequences of strains representing the spectrum of intratypic genetic diversity described to date identified two major evolutionary lineages designated phylogroups (PG) I, and II, and validated the early clustering of HAdV-E4 genomic variants into two major groups by low resolution restriction fragment length polymorphism analysis. In this study we expanded our original analysis of intra- and inter-PG genetic variability, and used a panel of viruses representative of the spectrum of genetic diversity described for HAdV-E4 to examine the magnitude of inter- and intra-PG phenotypic diversity using an array of cell-based assays and a cotton rat model of HAdV respiratory infection. Our proteotyping of HAdV-E strains using concatenated protein sequences in selected coding regions including E1A, E1B-19K and -55K, DNA polymerase, L4-100K, various E3 proteins, and E4-34K confirmed that the two clades encode distinct variants/proteotypes at most of these loci. Our in vitro and in vivo studies demonstrated that PG I and PG II differ in their growth, spread, and cell killing phenotypes in cell culture and in their pulmonary pathogenic phenotypes. Surprisingly, the differences in replicative fitness documented in vitro between PGs did not correlate with the differences in virulence observed in the cotton rat model. This body of work is the first reporting phenotypic correlates of naturally occurring intratypic genetic variability for HAdV-E4. IMPORTANCEHuman adenovirus type 4 (HAdV-E4) is a prevalent causative agent of acute respiratory illness of variable severity and of conjunctivitis and comprises two major phylogroups that carry distinct coding variations in proteins involved in viral replication and modulation of host responses to infection. Our data show that PG I and PG II are intrinsically different regarding their ability to grow and spread in culture and to cause pulmonary disease in cotton rats. This is the first report of phenotypic divergence among naturally occurring known genetic variants of a HAdV type of medical importance. This research reveals readily detectable phenotypic differences between strains representing phylogroups I and II, and it introduces a unique experimental system for the elucidation of the genetic basis of adenovirus fitness and virulence and thus for increasing our understanding of the implications of intratypic genetic diversity in the presentation and course of HAdV-E4-associated disease.

microbiology↗