Advancing dendrochronological studies of fire in the United States

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Dendroecology is the science that dates tree rings to their exact calendar year of formation to study processes that influence forest ecology (e.g., Speer 2010 [1], Amoroso et al., 2017 [2]). Reconstruction of past fire regimes is a core application of dendroecology, linking fire history to population dynamics and climate effects on tree growth and survivorship. Since the early 20th century when dendrochronologists recognized that tree rings retained fire scars (e.g., Figure 1), and hence a record of past fires, they have conducted studies worldwide to reconstruct [2] the historical range and variability of fire regimes (e.g., frequency, severity, seasonality, spatial extent), [3] the influence of fire regimes on forest structure and ecosystem dynamics, and [4] the top-down (e.g., climate) and bottom-up (e.g., fuels, topography) drivers of fire that operate at a range of temporal and spatial scales. As in other scientific fields, continued application of dendrochronological techniques to study fires has shaped new trajectories for the science. Here we highlight some important current directions in the United States (US) and call on our international colleagues to continue the conversation with perspectives from other countries

The North American tree-ring fire-scar network

Fire regimes in North American forests are diverse and modern fire records are often too short to capture important patterns, trends, feedbacks, and drivers of variability. Tree-ring fire scars provide valuable perspectives on fire regimes, including centuries-long records of fire year, season, frequency, severity, and size. Here, we introduce the newly compiled North American tree-ring fire-scar network (NAFSN), which contains 2562 sites, >37,000 fire-scarred trees, and covers large parts of North America. We investigate the NAFSN in terms of geography, sample depth, vegetation, topography, climate, and human land use. Fire scars are found in most ecoregions, from boreal forests in northern Alaska and Canada to subtropical forests in southern Florida and Mexico. The network includes 91 tree species, but is dominated by gymnosperms in the genus Pinus. Fire scars are found from sea level to >4000-m elevation and across a range of topographic settings that vary by ecoregion. Multiple regions are densely sampled (e.g., >1000 fire-scarred trees), enabling new spatial analyses such as reconstructions of area burned. To demonstrate the potential of the network, we compared the climate space of the NAFSN to those of modern fires and forests; the NAFSN spans a climate space largely representative of the forested areas in North America, with notable gaps in warmer tropical climates. Modern fires are burning in similar climate spaces as historical fires, but disproportionately in warmer regions compared to the historical record, possibly related to under-sampling of warm subtropical forests or supporting observations of changing fire regimes. The historical influence of Indigenous and non-Indigenous human land use on fire regimes varies in space and time. A 20th century fire deficit associated with human activities is evident in many regions, yet fire regimes characterized by frequent surface fires are still active in some areas (e.g., Mexico and the southeastern United States). These analyses provide a foundation and framework for future studies using the hundreds of thousands of annually- to sub-annually-resolved tree-ring records of fire spanning centuries, which will further advance our understanding of the interactions among fire, climate, topography, vegetation, and humans across North America

The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

<div><p>The hepatitis C virus (HCV) p7 protein is required for infectious virus production via its role in assembly and ion channel activity. Although NMR structures of p7 have been reported, the location of secondary structural elements and orientation of the p7 transmembrane domains differ among models. Furthermore, the p7 structure-function relationship remains unclear. Here, extensive mutagenesis, coupled with infectious virus production phenotyping and molecular modeling, demonstrates that the N-terminal helical region plays a previously underappreciated yet critical functional role, especially with respect to E2/p7 cleavage efficiency. Interrogation of specific N-terminal helix residues identified as having p7-specific defects and predicted to point toward the channel pore, in a context of independent E2/p7 cleavage, further supports p7 as a structurally plastic, minimalist ion channel. Together, our findings indicate that the p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production.</p></div

Mutation of the N-terminal helix is deleterious for infectious virus production <i>in vitro</i>.

<p><b>A)</b> Cartoon of the N-terminal tryptophan substitutions generated in J6/JFH p7. As in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.g001" target="_blank">Fig 1</a>, the secondary structure boundaries shown were previously deduced from p7 NMR data using HCV-J (genotype 1b) in 50% TFE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref041" target="_blank">41</a>]. <b>B)</b> HCV RNA levels in Huh-7.5 cells 8 and 72 hpe demonstrating that all p7 mutants replicate efficiently. J6/JFH lacking either p7 (Δp7) or the HCV glycoproteins (ΔE1E2) were used as additional assembly-defective controls. <b>C)</b> Infectious virus production quantified by limiting dilution assay on naïve Huh-7.5 cells shows that mutation of various residues in this region preclude generation of infectious HCV particles. <b>D)</b> Western blot analyses detecting HCV E2 antigen. Left panel: J6/JFH WT-, ΔE1E2-, E2-IRES-p7-, or p7-IRES-NS2-replicating Huh-7.5 cell lysates, used here to provide markers for E2p7NS2, E2p7, and E2 protein species. Right panel: Parallel western blot analysis comparing amounts of E2 relative to E2p7 between J6/JFH WT and N-terminal helix p7 mutants (positions 1–13).</p

Comparison of p7 structure models identifies the N-terminal helical region as a potential key modulator of p7 function.

<p><b>A)</b> Comparison of amino acid sequences and NMR secondary structural elements of p7 as determined by NMR in 50% TFE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref041" target="_blank">41</a>] (PDB entry, 2K8J), 125 mM DHPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref022" target="_blank">22</a>] (PDB entry, 2MTS), 100% MeOH [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref042" target="_blank">42</a>] (PDB entry, 3ZD0) and 200 mM DPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref047" target="_blank">47</a>] (PDB entry, 2M6X). The stars (*) indicate that additional amino acids (N-terminal FLAG tag and C-terminal polylinker) were fused to this construct. The green box highlights a region where secondary structural elements are quite divergent across models, especially between monomeric (first three sequences) and hexameric (bottom sequence) NMR-based models. h, helix; c, coil; t, turn. The sequence of the p7 (J6) used in this study is also shown for comparison. An amino acid similarity index is used where <i>asterisk</i> indicates invariant, <i>colon</i>, highly similar and <i>dot</i>, similar. <b>B)</b> Comparison of the NMR model in DPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref047" target="_blank">47</a>] and NMR/MD model in POPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref044" target="_blank">44</a>] showing the hexameric form and two opposing subunits in the hexamer. Lines shown in the left hand panels represent the membrane interfaces and hydrophobic core (between the middle two lines). The positions of both models relative to the membrane bilayer were deduced from MD simulations in a POPC bilayer as previously reported for model 1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref053" target="_blank">53</a>] and model 2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref044" target="_blank">44</a>]. N- and C-termini are noted by “N” and “C”, respectively. <b>C)</b> N-terminal helical packing in both models demonstrates similar packing and residues 9 and 12 in both models point towards the pore. Figures were generated from structure coordinates by using VMD (<a href="http://www.ks.uiuc.edu/Research/vmd/" target="_blank">http://www.ks.uiuc.edu/Research/vmd/</a> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref054" target="_blank">54</a>]) and rendered with POV-Ray (<a href="http://www.povray.org/" target="_blank">http://www.povray.org/</a>).</p

Identification of putative ion channel defective mutants by homology modeling and bafilomycin A1 rescue.

<p><b>A)</b> Molecular models of N-terminal region mutants that yielded >1 log reduction in infectious virus production compared to wild-type in a bicistronic context. The mutated residue Trp side chains are shown in red. Models provide insight into whether the mutation is likely to block the pore (e.g. H9W and S12W), disturb p7 intramolecular interactions (e.g. A10W), or interrupt p7 interactions with binding partners (e.g. A1W, A10W). <b>B)</b> Bafilomycin A1 rescue experiment schematic. Forty-eight hours post-electroporation, Huh-7.5 cells replicating control or p7 mutant viruses were supplied with cell culture medium containing bafilomycin A1 [8nM] or DMSO. Supernatants were collected 24 hours post-treatment, concentrated and dialyzed to remove excess bafilomycin A1, and then tittered on naïve Huh-7.5 cells to quantify infectious virus production. <b>C)</b> Resulting infectious virus titers from the experiment outlined in panel b. Mutant viruses yielding significantly more infectious virus production under bafilomycin A1 conditions compared with DMSO were identified using unpaired t-tests. Statistical results are indicated as follows: ns = not significant, * p<0.05, and *** p<0.0001. LOQ: lower limit of the limiting dilution assay. Black diamond (◆) indicates that these control viruses are monocistronic; all others shown are bicistronic. KRAA denotes J6/JFH with K33A and R35A mutations in p7.</p

Passage of deleterious N-terminal mutants identifies second site pseudo-revertants.

<p><b>A)</b> Coding changes identified within p7 by sequencing after passage of original p7 mutant genomes in Huh-7.5 cells. No virus was obtained after passage of H9W (indicated by the diamond). <b>B)</b> Western blot analyses detecting HCV E2 antigen. Left panel: J6/JFH WT-, ΔE1E2-, E2-IRES-p7- or p7-IRES-NS2-replicating Huh-7.5 cell lysates, used here to provide markers for E2p7NS2, E2p7, and E2 protein species. Right panel: Parallel western blot analysis comparing amounts of E2 relative to E2p7 between J6/JFH WT, original p7 mutant genomes and genomes harboring mutations identified after passage. <b>C)</b> HCV RNA levels in Huh-7.5 cells determined 8 and 72 hpe showing all p7 mutants (original and those harboring mutations identified after passage re-engineered into J6/JFH p7) replicate efficiently. <b>D)</b> Infectious virus in the supernatants quantified by limiting dilution assay on naïve Huh-7.5 cells demonstrating rescue of infectivity by mutations identified after passage re-engineered into the J6/JFH backbone.</p

Amino acid requirements at positions 6, 9, and 12.

<p>Infectious virus in the supernatants of monocistronic <b>(A, B & C)</b> or bicistronic <b>(D, E & F)</b> p7 mutants quantified by limiting dilution assay on naïve Huh-7.5 cells. Mutant viruses yielding significantly less infectious virus production as compared with wild-type were identified using unpaired t-tests. Statistical results are indicated as follows: * p<0.05, ** p<0.01 and *** p<0.001. Black diamond (◆) indicates that these control viruses are monocistronic. Note that the WT, GNN, Δp7, and ΔE1E2 titers reported in panels <b>E</b> and <b>F</b> are duplicated from panel <b>D</b> as these viruses were all analyzed in parallel.</p