Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods, and nematodes. Though initially described as simple single-gene nonsegmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as “ambigrammatic” harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2-kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8-kb “Robin” RNA. We developed a system to launch CxNV1 in a naive mosquito cell line and then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively translating ribosomes on host mRNA or coinfecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission.IMPORTANCE Fundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand. Saccharomyces cerevisiae and in nematodes, transmission is thought to occur via cytoplasmic inheritance during horizontal transfer in yeast or cell division, including animal germ lines, without any extracellular virion  found in the reverse direction overlapping the RdRp ORF and spanning almost the entire length of the segment. Such narnaviruses have hence been described as “ambigrammatic,” as both the forward and reverse ORFs have the potential to be translated. This is achieved by avoidance of the codons UUA, CUA, and UCA, which encode stops in the −0 frame , 12. TheOverlapping ORFs are common among viruses. Advantages include compact genomes, novel genes, and translation regulation, at the expense of evolutionary constraints imposed on the sequence by coding in two frames instead of one . AmbigraThese compelling observations motivate our investigation of CxNV1, an ambigrammatic narnavirus found in wild-caught mosquitoes. Its 3.2-kb RdRp-encoding RNA is associated with a second RNA, the 0.8-kb “Robin” segment. Both segments are ambigrammatic. Here, we analyzed the CxNV1 genome in a persistently infected cell line, developed a launching system to test the requirement for the rORF, examined rORF translation by mass spectrometry, and interrogated the association of ribosomes with each viral RNA by ribosome profiling.Culex tarsalis mosquito (https://doi.org/10.7272/Q6GX48SV]). Meta-transcriptomic next-generation sequencing (mNGS) revealed no evidence for other cellular organisms in the cell line, confirming Culex tarsalis as the host species . An end-specific adapter-ligation sequencing approach revealed complementary ends on each CxNV1 segment, beginning with 5′-GGGG and ending with CCCC-3′ with a 21 nucleotide (nt) 3′ hairpin , another persistent coinfection in CT cells, mNGS clearly showed a large increase in the coverage of the 3′ untranslated region (UTR) , consistent with known subgenomic flaviviral RNAs . In summNext, we sought to determine whether DNA forms exist for CxNV1, which belongs to a family classified as +ssRNA viruses but infects insect cells which commonly endogenize viral sequences . As expeOverall, these analyses support the bi-segmented nature of CxNV1 as an RNA virus with substantial negative-strand contribution. Importantly, the complete and accurate genomes allowed us to attempt to launch the virus in uninfected cells.Culex quinquefasciatus mosquito . While a single nonsynonymous mutation in the rORF opposing RdRp had no effect (“ns1”), the introduction of a single stop codon in the rORF at the same position (“stR1”) resulted in loss of the RNA by 2 weeks posttransfection . LikewisThe requirement for an intact rORF in the essential RdRp segment implies translation of the full-length rORF or other interaction with ribosomes. Mass spectrometry was used to investigate translation in persistent CxNV1 infection. Peptides were detected from across the entire length of both forward and reverse ORFs on the RdRp segment -digested monosome fraction of CT cells with either cycloheximide (CHX) or no treatment (no-drug) before lysis (Table S7). RNAseq libraries were prepared from total RNA (totRNA) of the same samples using an mNGS workflow. As the additional pretreatment had little effect, the four libraries were considered near replicates.de novo assembled for the following analyses, as no genome is currently available for Culex tarsalis . Among these host transcripts, a consistent ratio of footprint to total RNA reads was observed . However, footprints on CxNV1 showed several unexpected features. First, the abundance of footprints relative to total RNA abundance was substantially lower for CxNV1 than for host transcripts , Fig. S9We performed a series of additional analyses to determine whether technical factors could explain these observations. With regard to sequencing, insufficient sampling as a source for CxNV1-specific footprint patterns was ruled out, as an ample number of footprint reads were observed for both CxNV1 segments, and low-abundance host transcripts such as PP2A did not show the plateau pattern. PCR jackpotting was also considered a potential confounder. Removing PCR duplicates by collapsing identical footprint + unique molecular identifier (UMI) sequences caused greater depletion of CxNV1-mapping footprints than other genes . However, the extreme concentration of CxNV1 footprint peaks could exceed the diversity in a random 5-nt UMI library. Regardless, the CxNV1-specific plateau pattern was retained after duplicate collapse.Next, steps upstream of sequencing were examined. Sequence biases inherent to multiple steps in sample preparation, including nuclease digestion, adapter ligation, and circularization, are known to affect footprint recovery , 22. Forr] and P value for RdRp, r = 0.02, P = 2.0e-1; Robin, r=−0.14, P = 2.8e-5). In summary, the plateau pattern of CxNV1 footprints was not solely attributable to abundant viral RNA or antiviral pathways.We examined whether the unusual CxNV1 footprints could be attributable to abundant viral RNA or antiviral pathways, RNA secondary structure, or features at the amino acid level. Footprint data for CxNV1 were first compared to other persistent viruses in the cell line, including CALV and the negative-sense, tri-segmented Phasi Charoen-like phasivirus (PCLV). For CALV and PCLV, relatively uniform footprint coverage was observed across the canonical ORFs with expected drop-offs in the 3′ UTRs . To assess whether the CxNV1-specific footprints represent nonspecific interactions with abundant RNAs, footprints on the genomic negative strand of PCLV were examined, as an example of an abundant RNA that is likely not translated. The negative-strand PCLV footprints were less abundant than positive-strand footprints , did not show the CxNV1-specific plateau pattern , and had length distributions that were significantly different from the reference EF1a positive-strand distribution , suggesting that the negative-strand reads for PCLV represented a different process than CxNV1 footprints. Noncoding host transcripts for additional comparison could not be confidently identified given the lack of existing genome for this species. Lastly, the distribution of footprints in our ribosome profiling was related to mapping of small RNAs previously sequenced from the same cell line by comparing coverage profiles. No direct correlation or consistent pattern was observed for CxNV1  , 25. To Amino acid features were also considered. In yeast, codons encoded by less abundant tRNAs tend to be translated more slowly . Due to 27–Finally, polysome profiling was performed to ascertain ribosome density on CxNV1 RNA. Reverse transcription-quantitative PCR (RT-qPCR) targeting CxNV1 and GAPDH was performed on fractions from the polysome gradient of undigested RNA, with or without EDTA treatment of lysates before gradient separation to release RNA-bound complexes including ribosomes  is a relatively new and unexplored finding. Our data suggest that CxNV1 RdRp is required for replication of the Robin RNA. While the RdRp segment persisted without Robin in cell culture, this RdRp-only state has not been observed for CxNV1 in wild-caught mosquitoes . It is pThe relationship between the RdRp and Robin segments of CxNV1 may hinge on the shared feature of overlapping ORFs on opposite strands. Our experiments demonstrated that premature truncation of the rORF on the RdRp segment resulted in loss of the RNA, indicating a requirement for the presence of the rORF. As for the importance of the rORF protein sequence, we note that the forward ORFs are considerably more conserved than the rORFs, as shown in a previously published data set of related CxNV1 sequences , where tWhat, then, is the role of the essential overlapping opposite-sense ORFs? While the rORF appears to be translated, the bulk of viral RNAs appear to be devoid of ribosomes. Assuming that the footprints observed in the ribosome profiling libraries truly indicate ribosome-protected RNA, then the interaction of ribosomes with CxNV1 is unlike typical host RNA translation. We propose a model where a small fraction of CxNV1 RNA is occupied by regions of densely packed ribosomes . This co37–We can only speculate on how ribosome-coating of viral RNAs might contribute to viral fitness. Perhaps ribosomes fill roles typically performed by nucleocapsid proteins in other viruses, such as protecting the viral RNA from host cell degradation, maintaining RNA in a polymerase-accessible state, or influencing transmission of viral RNA during cell division. Further study of CxNV1 RNA stability in the context of various deficiencies in host cell antiviral pathways may be informative.Alternative interpretations of the data, including technical caveats, were considered, but these were found to be insufficient to explain the observations. We considered and rejected artifacts from PCR and library preparation steps and bias in nucleotide and amino acid sequence. Conceivably, the footprints might represent contaminating RNA or protection by a nonribosomal RBP found at the same sucrose density as the monosome peak. The unusual footprint size distribution for CxNV1-mapping footprints warrants consideration. On one hand, nonconforming size distributions are often used to exclude regions lacking bona fide translation activity, as in many noncoding RNAs and 3′ UTRs . For unu42–In conclusion, this investigation suggests a previously unappreciated strategy among viruses, whereby a small fraction of the RNA from this unique narnavirus exists in a state characterized by bound, densely packed, nonprocessive ribosomes. This may represent a novel mechanism by which an RNA virus can coopt the host translational machinery to facilitate persistent infection and transmission in the absence of an intact virion and extracellular phase.Culex tarsalis embryos (CT) and Culex quinquefasciatus ovaries (Hsu) were a generous gift of Aaron Brault  (Drosophila medium (Gibco) supplemented with 10% vol/vol fetal bovine serum at 28°C in room air, passaged by scraping, and tested negative for mycoplasma monthly.Established cell lines derived from CO, USA) , 48. Celde novo assembly, and identification of viral sequences . After adding External RNA Control Consortium (ERCC) RNA spike-in mix (Life Technologies), the sequencing library was prepared using a NEBNext Ultra II directional RNA library prep kit (New England Biolabs [NEB]) and sequenced with a paired-end 150-bp run on an Illumina NextSeq instrument. The raw reads were processed via the IDSeq pipeline  for initial processing, including quality filtering, deduplication, host subtraction, equences . These fequences .TATGCA followed by 0 to 5 Ns before the Illumina TruSeq Adapter 5 (TSA5) sequence to allow for dephasing during read1 sequencing (oHR546/547/548/549/550/551). For the RNA ligation method, these ligation oligos were ordered with 5′ phosphorylation and 3′ blocking with C3 Spacer (IDT), pooled in equimolar ratios, preadenylated at the 5′ end using Mth RNA ligase (NEB), and then ligated to total RNA extracted from CT cells using 5′ App DNA/RNA ligase (NEB). Next, a TSA5-complementary oligo (oHR552) was used to initiate reverse transcription with SSIII (Thermo Fisher) followed by basic RNA hydrolysis and column purification . Then, the regions of interest were amplified and barcoded using nested PCR with Phusion high-fidelity DNA polymerase (NEB), an internal primer containing TSA7 and a footprint sequence ∼100- to 150 nt from the predicted end , and external primers containing the P5/P7 and i5/i7 Illumina adapter and index sequences.Two strategies were used to identify the ends of the CxNV1 RNA, ligation to RNA (for the 3′ end) and reverse transcription and then ligation to cDNA (for the 5′ end). See Table S9 for oligo sequences. For both strategies, the oligo ligated to the unknown end contained a handle For the cDNA ligation method, targeted reverse transcription was performed on the extracted CT cell RNA with the footprint oligos oHR542/543/544/545 and SSIII (Thermo Fisher), which is known to add a few nontemplated bases after reaching the 5′ end of the RNA with a preference for cytosine . The pooFor analysis of ends libraries, reads were first quality filtered using PriceSeqFilter with the flags -rqf 85 0.98 -rnf 90 and then trimmed to the read beyond the handle TGCATA. Reads were then aligned to the consensus sequence for the region of CxNV1 well supported by >4 reads from mNGS sequencing (see above) using Bowtie2 (v2.2.4). Variants in the terminal 8 bases were analyzed to determine the most likely consensus sequence, taking into account potential biases from reverse transcription and ligation and potential biological variation. RNA folding of terminal sequences was performed using the RNAstructure web server with temperatures according to narnavirus host species: 28°C for insect, 30°C for yeast .Experiments to determine whether CxNV1 has DNA forms were initiated by extracting RNA from CT cells using a Direct-Zol RNA MicroPrep kit (Zymo Research) and, separately, DNA from matched aliquots of CT cells using a Quick-DNA miniprep kit with proteinase K (Zymo Research). As a control, RNA and DNA were also extracted from Hsu cells. Extracted RNA was treated with DNase during column purification, and extracted DNA was treated with RNaseA (Thermo Fisher) and/or with DNase (Invitrogen) as a control. Amplification reactions were performed using KAPA SYBR FAST one-step qRT-PCR (Roche), with or without reverse transcriptase, on the purified RNA or DNA spiked with a Neo-containing plasmid, with 35 cycles, 60°C annealing temperature, 30 sec extension, and the following primer pairs: EF1a oHR561/565 (intronic) and oHR672/673 (exonic); CxNV1 RdRp oHR504/509, oHR502/503, and oHR513/514; CxNV1 Robin oHR653/654, oHR538/539, and oHR540/541; and Neo oHR691/692 (Table S9). PCRs and agarose gel electrophoresis to analyze their products were run in parallel for all conditions. To determine the identity of faint bands at similar size to target products, reactions were rerun with 40 cycles, and then bands were cloned using Topo TA cloning (Thermo Fisher) and Sanger sequenced.SRR8668667 and SRR8668668) were downloaded, adapter sequences were trimmed as needed using Trimmomatic (v0.39) with Illumina TruSeq smallRNA adapters and flags ILLUMINACLIP:$adapter_fasta:1:0:0:1 MINLEN:18, quality filtered using PriceSeqFilter with flags -rqf 85 0.98 -rnf 90, and then aligned to the combined CT host plus virus transcript reference  using Bowtie (v1.2.3) with flags -v 1 -k 1 -m 1.Small RNA sequencing of CT cells with or without infection with West Nile virus (WNV) was previously performed by Göertz et al. . The datPRJNA605178) were aligned to the assembled contigs for each sample for contigs assigned to viral species by the original study authors. The ratio of concordantly aligned read pairs derived from positive (coding) versus negative strand was calculated. Viral segments with <100 concordantly mapped read pairs or <3 samples  were discarded from the analysis.mNGS of wild-caught mosquitoes in California was previously performed by Batson et al. . QualityTo clone CxNV1 from CT cell RNA, an iterative process of RT-PCR  and topo cloning (Invitrogen) of long genomic fragments was performed, ultimately completed with primers oHR594/593, oHR592/586, oHR585/589, and oHR619/620 for 4 fragments of RdRp and with oHR621/622 and oHR623/624 for 2 fragments of Robin, each flanked by BbsI or BsaI cut sites (Table S9). The fragments were assembled into a pUC19 vector using In-Fusion cloning (TaKaRa Bio) with inverse PCR to add ends determined by the ligation sequencing method described above, ultimately generating plasmids pHR96 (RdRp) and pHR97 (Robin), which contain SmaI-flanked full-length genomic RNAs.Mutant versions of RdRp and Robin were generated with a combination of PCR, GeneBlocks (IDT), restriction enzyme, and In-Fusion cloning. To control for effects of mutations at the RNA level, mutants with stop codons were compared to mutants with nonsynonymous changes at identical nucleotide positions. Unique restriction enzyme sites were introduced to distinguish plasmids easily. Details can be found in Table S5.To generate plasmids for expression of CxNV1 RNAs in insect cells, a backbone was first created by inserting the T7-HDR (hepatitis D ribozyme) region from p2RZ (gift from Kristeene Knopp) into pIEX-4 (gift from Wesley Wu) and then inserting this cassette into pAc5-STABLE1-Neo , generating “empty” plasmid pHR106. The RdRp or Robin segments were inserted into this backbone, generating pHR107 and pHR108, respectively, each containing separate Ac5-GFP and hr5/IE1-narnavirus expression constructs. In these plasmids, narnaviral RNA is transcribed with ∼72 nt upstream of the viral 5′ terminus and with the hepatitis D ribozyme to cleave at the viral 3′ terminus. In addition, the green fluorescent protein (GFP) was swapped for mCherry in expression plasmids containing CxNV1-Robin.To launch CxNV1 in a naive cell line, Hsu cells were transfected using X-tremeGENE HP DNA transfection reagent (Roche) with plasmid(s) expressing a fluorophore under the control of the Ac5 promoter, and the CxNV1 RdRp, Robin, mutants, or empty under the control of the IE1 promoter with hr5 enhancer, as described above. In initial experiments, both segments were in GFP-expressing vectors; subsequent experiments were performed with Robin in a backbone expressing mCherry instead of GFP, showing that when multiple plasmids were transfected concurrently, >95% of cells received either both or neither plasmid, and yielding no difference in results. As antibiotic-based selection was inefficient, cells were first sorted for successful transfection at 2 to 6 days posttransfection (fluorophore-positive) and then passaged and countersorted for loss of plasmid at 10 to 12 days posttransfection (fluorophore-negative) using a Sony SH800S sorter. At time points from 2 to 9 weeks posttransfection, cells were collected for nucleic acid extraction (including DNase treatment for RNA extractions) and PCR amplification. To determine persistence of viral RNA after loss of plasmid DNA, RT-PCRs were performed with the following primer pairs: oHR672/673 (EF1a), oHR513/514 (CxNV1 RdRp), oHR653/654 (CxNV1 Robin), and oHR691/692 (Neo) (Table S9).g at 4°C and flash-freezing the supernatant. The protein concentration was measured using the Bradford assay (Bio-Rad). Lysates were diluted in 2× sample buffer  supplemented with 10% vol/vol beta-mercaptoethanol, boiled at 95°C for 3 min, sheared with a 26G needle, boiled 2 min, and centrifuged, and then 15 μg was loaded onto a NuPAGE 4 to 12% Bis-Tris polyacrylamide gel (Invitrogen) and separated by electrophoresis. Gel bands were cut from two regions, 100 to 150 kD and 25 to 37 kD, and processed via trypsin digestion and liquid chromatography tandem mass spectrometry (LC-MS/MS) as detailed below. Coomassie stain of parallel gels confirmed adequate separation of the complex lysate. Biological duplicates were performed 10 months apart.Lysates were prepared from two 15-cm tissue culture plates of CT cells by lysing in ice-cold radioimmunoprecipitation assay (RIPA) buffer  with cOmplete EDTA-free protease inhibitor cocktail (Roche) and then rotating overhead for 10 min at 4°C, followed by centrifuging for 10 min at 16,000 × Mass spectrometry was performed by the Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at the University of California at Berkeley (UC Berkeley). A nano LC column was packed in a 100-μm-inner diameter glass capillary with an emitter tip. The column consisted of 10 cm of Polaris c18 5-μm packing material. The column was loaded by use of a pressure bomb and washed extensively with buffer A (see below). The column was then directly coupled to an electrospray ionization source mounted on a Thermo Fisher LTQ XL linear ion trap mass spectrometer. An Agilent 1200 high-pressure liquid chromatography (HPLC) system equipped with a split line so as to deliver a flow rate of 300 nL/min was used for chromatography. Peptides were eluted with a 90 minus gradient to 60% B. Buffer A was 5% acetonitrile/0.02% heptafluorobutyric acid (HBFA); buffer B was 80% acetonitrile/0.02% HBFA.Culex quinquefasciatus and custom viral databases supplemented with sequences of common contaminants. The database was concatenated to a decoy database in which the sequence for each entry in the original database was reversed  using ProLuCID/Sequest, DTASelect2, and Census –57. Tand–reversed . LTQ datreversed , 56 with2, 1 mM dithiothreitol [DTT], and 100 μg/mL CHX, supplemented with 1% vol/vol Triton X-100 and 25 U/mL Turbo DNase I [Thermo Fisher]). Clarified cell lysates were treated with micrococcal nuclease (MNase) to digest RNA not protected by ribosomes. MNase has previously been effective for footprinting insect cells  at 37°C for 2 min. Ribosome-protected footprints from cycloheximide-pretreated and no-drug samples were prepared for sequencing as described in a recently updated protocol for ribosome profiling of mammalian cells, with slight modifications . Brieflyct cells , and it ct cells . In our The 3′ ends of the ribosome footprint RNA fragments were then treated with T4 polynucleotide kinase (NEB) to allow ligation of a preadenylated DNA linker with T4 Rnl2(tr) K227Q (NEB). The DNA linker incorporates sample barcodes to enable library multiplexing, as well as unique molecular identifiers (UMIs) to enable removal of duplicated sequences. To separate ligated RNA fragments from unligated DNA linkers, 5′-deadenylase (Epicentre) was used to deadenylate the preadenylated linkers, which were then degraded by the 5′-3′ ssDNA exonuclease RecJ (NEB). After rRNA depletion using the Ribo-Zero Gold rRNA removal kit (Illumina), the RNA-DNA hybrid was used as a template for reverse transcription with Superscript III (Thermo), followed by circularization with CircLigase (Epicentre). Finally, PCR of the cDNA circles attached suitable adapters and indices for Illumina sequencing and then libraries were quantified and pooled. The library was sequenced with a single-end 50-bp run on an Illumina HiSeq 4000 instrument.The corresponding RNA-seq samples were prepared from total RNA of the same cell lysates. RNA was extracted using a Direct-Zol RNA MicroPrep kit (Zymo Research), and then libraries were prepared using a NEBNext Ultra II directional RNA library prep kit (NEB) and, finally, sequenced with a paired-end 150-bp run on an Illumina NextSeq instrument.Culex tarsalis. The closest species, Culex quinquefasciatus, is homologous enough in exonic regions for credible alignments of 150-nt paired-end reads, but we found noncoding regions including introns to be dissimilar enough to make genome-wide alignments unreliable and annotations somewhat lacking. Instead, we chose to assemble a set of transcripts likely to be well conserved and spanning from low to high abundance, namely, genes for elongation factors, initiation factors, and ribosomal proteins, and a few likely single-copy canonical genes, EF1a, GAPDH, HSP70, RPS17, and PP2A. We abandoned genes where the annotations were unclear and which may have multiple copies in the genome. We first extracted mRNA sequences for genes of the classes listed above from the CulPip1.0 genome assembly tool (NCBI). We then aligned the quality-filtered reads from the 150-nt paired-end sequencing of the parental CT cell line. For transcripts with promising homology, we extended the contig into 5′ and 3′ UTRs using PriceTI (v1.2) and then validated by remapping paired-end reads and, finally, selecting transcripts with unambiguous mapping at >4 read coverage. See Table S8 for BLAST results of final host reference transcripts.No genome reference was available for The genomes for persistent RNA viruses in the CT cell line were generated by aligning 150-nt paired-end reads to the contigs assembled with SPAdes in the IDSeq pipeline for error correction and contig extension. Culex narnavirus 1 (CxNV1), Calbertado virus (CALV), and Phasi Charoen-like phasivirus (PCLV) were assembled this way. Anomalies in the alignments and assembly of Flock House virus (FHV) were suspicious for defective viruses or other multiplicity, so FHV was removed from consideration.For processing of ribosome profiling data, linker sequences were removed, and samples were demultiplexed using FASTX-clipper and FASTX-barcode-splitter (FASTX-Toolkit v0.0.13). To generate quality and duplication metrics for the entire library, reads were processed with FASTQ-quality-filter with flags -Q33 -q 37 -p 80, then trimmed to remove the 3′ sample barcode (4 nt) and a single base of low quality at the 5′ end (1 nt), and then processed with cd-hit-est (v4.8.1) with flag -c 1 to require 100% identity. For all other analyses, a preliminary alignment of sample barcode- and UMI-trimmed reads to the reference was performed with Bowtie (v1.2.3) and flags -v 1 -k 1 -m 1 to allow a single mismatch; then, the original sequences were retrieved, sample barcodes were trimmed, and quality filtering was performed as described above. At this stage, reads were optionally collapsed on 100% identity of the read+UMI using cd-hit-est as above, and then the UMIs were trimmed. From this step forward, parallel pipelines were used for the collapsed and uncollapsed reads. Reads were finally mapped to the reference using Bowtie as above, with SAMtools (v0.1.19) to generate sorted, indexed BAM files.Culex tarsalis mitochondrial genome  with flags -rqf 85 0.98 -rnf 90 and then mapped to the reference using Bowtie2 (v2.2.4) with default parameters. From these BAM files, quantification of reads within the CDS on each strand was performed using Bedtools coverage, and coverage at each nucleotide position was determined using Bedtools genomecov with flag -d. Separate mapping to the reference transcripts plus custom n et al. ) plus ERCustom scripts were used for further analysis and plotting in Python 3.8, including a combination of plastid (v0.5.1) , biopythFor periodicity analysis, footprints were first mapped to the 5′ end using plastid with –fiveprime –normalize and then scaled within each refseq+sample pairing to be comparable across samples. Selecting the CDS region with 25-nt padding from either end, the power spectral density was estimated using Welch’s method performed by scipy.signal.welch with parameters window=‘hann’, nperseg=500, noverlap=250, scaling=‘density’, average=‘median’.For modeling the impact of nucleotide bias on footprint position, we first developed a footprint generator as follows: choice of 5′ cut site on a given sequence using numpy’s random.default_rng function with probabilities based on the empirical observations of bases found at the positions on either side of the 5′ cut site; then choice of 3′ cut site based on similar choice with base-preference weighting at the 3′ end in addition to weighting by empirical length distribution. This generator was run to produce 10,000 footprints from both the actual and a scrambled CxNV1 RdRp sequence and then was repeated for a total of 10 runs. The coverage of modeled footprints in each scenario was then compared to the observed coverage using Pearson correlation.For RNA secondary structure analyses, RNAfold (v2.4.0) from the ViennaRNA package was used to calculate minimum free energy along sliding windows of the stated sizes, with a temperature of 28°C .x – average), where MFEx is the MFE at position x and win is the window size, and then compared to the numpy gradient function of the footprint profiles .For analyses of footprint context (RNA secondary structure and amino acid features), a window of adjacent nucleotides/amino acids was first selected. For amino acid analysis, this window included 20 residues upstream of the codon at 2/3 of the distance from the 5′ end of the footprint, roughly corresponding to residues likely to be within the ribosome exit tunnel . FeatureCT cell lysates were prepared and separated on 10 to 50% mass/vol sucrose gradients as for ribosome profiling above, except without any RNase digestion step. In addition, the lysate was separated into a control condition or treated with 30 mM EDTA and then incubated on ice for 5 min prior to loading onto sucrose gradients. Fractions were then collected using the Biocomp Gradient Station for the full volume that could be sampled, which excludes the small amount of residual volume of densest sucrose and any pelleted material.in vitro transcribed RNA (generated by T7 transcription off a Firefly luciferase [Fluc]-containing plasmid). RNA extraction was performed in 96-well format on a Bravo automated liquid handling platform (Agilent) using the Quick-DNA/RNA viral MagBead kit (Zymo Research) with proteinase K and DNase steps. RT-qPCR was then performed in 384-well format with a Luna universal probe one-step RT-qPCR kit (NEB), assaying Fluc , GAPDH (oHR720/721/722), CxNV1 RdRp (oHR729/730/731), and CxNV1 Robin (oHR738/739/740), in triplicate wells for each RNA-target gene combination (Table S9).An aliquot of each fraction was then mixed 1:1 with 2× DNA/RNA Shield (Zymo Research) spiked with qC) values using empirical amplification efficiencies for each primer/probe set and the delta-delta threshold cycle (TC) method to normalize by Fluc. For each target gene, the relative RNA quantities in each fraction were then normalized across all fractions sampled in the gradient. For each lysate/treatment combination, 2 to 3 technical replicates were run on separate sucrose gradients and subsequent steps. A total of 2 biological replicates of cell lysates were collected for the non-EDTA condition.Standard analysis was performed on quantification cycle (MW226855 and MW226856. Raw NGS data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject number PRJNA675022. Raw mass spectrometry data have been deposited at ProteoSAFe (massive.ucsd.edu) under accession number MSV000086532. Supplementary materials are available at https://doi.org/10.7272/Q6GX48SV.The complete genome sequence of CxNV1 is available at GenBank under accession numbers


Pulau Tioman is a famous tourist island off Peninsular Malaysia with beautiful coral reefs. This study aims to assess the health of the coral reefs surrounding Pulau Tioman based on the application of the Foraminifera in Reef Assessment and Monitoring Index (FI). Ten sampling sites around Pulau Tioman were studied with a total of 30 samples. Eight orders, 41 families, 80 genera, and 161 species of benthic foraminifera were identified. The agglutinated type of foraminifera constituted 2–8% of the total assemblages. Calcareous hyaline and porcelaneous groups represented 79% and 19% of the total assemblages, respectively. Symbiont-bearing taxa were the most common foraminifera. The results indicate that most of the sampling sites are conducive for coral reef growth with good recoverability from future stress to the ecosystem. However, several areas with higher coastal development and tourism have reduced water and sediment quality. Therefore, the limit on the number of visitors and tourists should be revised to enable coral growth and health. The FI values in this study showed a positive correlation with good water qualities and a negative correlation with organic matter enrichment. The FI is a good measure to assess the health of a coral reef and can be applied to other reef ecosystems around Malaysia. Coral reefs provide goods and services to marine tropical and subtropical regions2. Globally, coral reef ecosystems are threatened or have disappeared due to climate change and human intervention3. On a larger scale, elevated sea temperatures and ocean acidification caused by climate change have challenged the natural resilience of tropical reefs4. Local stressors from human activities, such as coastal development and pollution, reef predatory practices, and diseases, usually reduce the potential of reef recovery from the ill effects of climate change6. Because coral reef ecosystems provide various natural environmental services, their ability to survive the global climate anomaly is important. Consequently, understanding the health status of local individual reefs is important to ensure that the coral reef survives and recovers from likely mass mortality events4.The coral reef ecosystem is among the most biologically diverse ecosystems in the world that plays a vital role in shaping the balance of environmental processes over the past 200 million years9. Benthic foraminifera have been proven to be excellent indicators of sediment quality, heavy metal pollution, organic pollution, and water quality13. Many taxa of Unicellular foraminifera are abundant in reef ecosystems; some of these taxa have similar ecological requirements as those of reef building corals11. The development of the Foraminifera in Reef Assessment and Monitoring (FORAM) Index (FI) by Hallock et al.11 has enabled continuous monitoring of the health of coral reefs. The FI was developed as a low-cost monitoring tool to indicate whether the quality of water surrounding a reef ecosystem can support reef growth11. This index was first applied in the Western Atlantic reef11 and has subsequently been applied widely in other regions, including the Great Barrier Reef in Australia8, reefs in Brazilian waters14, and the Saronikos Gulf, Greece15. The reliability, simplicity, and cost effectiveness of the FI has made it a suitable indicator for the monitoring of coral reef.Benthic foraminifera can successfully be used to monitor the potential recovery of the reef ecosystemMonitoring the environmental health of the surrounding reef is very important for natural park management authorities to safeguard the coral ecosystems and maintain their ecological importance in Pulau Tioman. This study aims to assess and monitor the health of the reef environment surrounding the Pulau Tioman island based on the application of the FI.Amphistegina lessonii (average of 22%) and the least dominant species (< 4%) were Bolivina vadescens, Elphidium neosimplex, Heterolepa dutemplei, Heterolepa subhaidingerii, Mikrobelodontos bradyi, Milliolinella suborbicularis, Operculina discoidalis, Parahourinoides fragillissimus, Quinqueloculina incisa, Quinqueloculina sulcata, Triloculinella bertheliniana, and Triloculinella parisa. Overall, the agglutinated foraminifera contributed 2–8% of the total foraminifera assemblages in Pulau Tioman. Calcareous hyaline and calcareous porcelaneous groups represent on average 79% and 19% of the total assemblages, respectively   varied between 4.5 and 6.9 mg/L with the highest amount of DO recorded at B1 (Kampung Mukut). Meanwhile the water pH recorded was between 8.1 and 8.7.The first and second axes of the Principal Component Analysis (PCA) explained 58.38% of the variations between the water quality and sediment quality variables Fig. . In addip < 0.05). The axis-1 of Canonical Correspondence Analysis (CCA) explained 29.1% of the variance between the species and environmental variables  and axis-2 explained 21.8%  and Assilina ammonoides (22%). Group B consisted of Stations E3, F2, G1, and F1. The species that dominated the assemblages in this group was Ammonia tepida with an average relative abundance of 17%. Group C consisted of Stations D1, H2, and H3, and was dominated by Calcarina gaudichaudii (34%) and Amphistegina lessoni (23%). Group D consisted of the remaining 21 stations . The common species identified for the Group D area were Amphistegina lessoni (27%) and Amphistegina papillosa (8%).The Monte Carlo permutation test showed that the foraminifera species are linearly related to the water quality and sediment quality data  than those reported from the fringing reef environment in Brazil19. Amphistegina lessoni and Calcarina gaudichaudii, which are among the most widespread species found in Indo-Pacific waters17, are highly abundant and common in Pulau Tioman. Amphistegina lessoni, for instance, occurs at all stations around Pulau Tioman, except in F3 (Mesoh), where conditions of high water depths (> 18 m), algal distribution, and turbidity exist, which may restrict their presence20. Despite relatively lower abundances (4–55%) compared to that reported in the study on the northern atoll in the South China Sea21, the calcareous porcelaneous group  contributed to > 50% of the total foraminiferal assemblage in Pulau Tioman. In Mesoh, where the water depth was > 15 m and slightly murky, there was a significant increase in porcelaneous representatives, such as Lachlanella compressiostoma (35%), Triloculina tricarinata (23%), and Triloculinella chiastocytis (21%). This finding supports the observations reported in Uthicke et al.8 in the Great Barrier Reef, where a high abundance of miliolids was recorded in turbid waters. Triloculina was common in the Dongsha Atoll in the northern South China Sea, but their abundance was recorded to be < 20%21.The foraminifera assemblages in Pulau Tioman are dominated by rotaliid genera, such as Appendix , which iAmphistegina and Calcarina are common in reef environments worldwide23, especially in Southeast Asia25. Most living Amphistegina can be found attached to reef substrates, with less of a presence in sediment, and usually occur in high abundance in coral reef environments9. Owing to their ecological requirements and distribution, foraminifera assemblages are among the best candidates to monitor reef health. Opportunistic taxa, especially Ammonia, Elphidium, and Bolivina, are among those that are tolerant to environmental variations13. Hence, typically polluted marine conditions, with organic matter enrichment and reduced dissolved oxygen, allow these groups to opportunistically increase in abundance15.Calcarina spp., correlated well with the increase in the dissolved oxygen concentration, salinity, and pH. Previously, Prazeres et al.26 suggested the need for calibration of the FI for the Indo-west Pacific where Calcarinidae occur. This is because Calcarina spp. usually thrive even in mesothrophic reefs20. Despite this concern, our study indicated that not only Calcarina spp. exhibits a good correlation with the water quality. Their relative abundance was significantly higher (> 15%) in Salang Bay, an area adjacent to where Shahbudin et al.3 reported the highest percentages of live coral coverage. Therefore, based on the species-environment relationship assessment, we speculate that calibration of the FI was not necessary for our study area.In addition, the CCA indicated that environmental variables, such as the concentration of dissolved oxygen (mg/L), pH, salinity, and organic matter composition, had the most significant effect on the distribution of benthic foraminifera around Pulau Tioman Fig. . Symbion3. Additionally, active tourism related activities, such as trampling by divers or snorkelers and resuspension of sediment by boats has increased the mortality rate of corals28.The increase in construction and land reclamation activities around the east coast region has exposed many coral reef areas to high rates of sedimentation and a consequent reduction of the diversity of live corals including those in Pulau Tioman14. Despite the excellent performance of the FI in numerous studies30, Prazeres et al.26 have raised some concerns on the application of this index in new study areas. Therefore, to address this concern and reduce the bias associated with the application of the FI index, we performed PCA. Based on the PCA results  that recorded lower FI values (FI < 4) in this study are famous diving and snorkeling sites, and their reef conditions are similar to those established by Shahbudin et al.Numulites venosus and Operculina ammonoides. Both symbiont-bearing species that belong to the Nummulitidae family have been reported to have better growth rates in regions with low light34, thereby explaining their increase in abundance at the deeper reef slope area (> 18 m water depth) in west Pulau Tioman. The calculated FI for Group A (FI > 6) shows that the water quality conditions within these stations (F3 and G3) can be classified as oligotrophic, which is optimal for reef recovery26. Hence, despite the intensity of diving activity in Mesoh and Panuba Bay, the increased distance from the shoreline has reduced the anthropogenic impacts on coral reefs and supported healthy coral growth and recovery35. Meanwhile, Group B recorded a significant increase in the porcelaneous foraminifera group, with a higher abundance of stress-tolerant taxa, such as Ammonia tepida. In west Pulau Tioman, the high average composition of organic matter (3.81%) in the sediment serves as a food source for heterotrophic taxa and increases the number of stress tolerant species, such as Ammonia. The FI values for this group varied between 2.7 and 4.0, with the lowest values recorded in the vicinity of Mesoh, where high snorkeling and diving activities occur. The sheltered beach in Mesoh is among the most popular sites for locals and tourists to enjoy swimming and snorkeling. Based on the observations during field sampling, the water in F1 (Mesoh) is more turbid with numerous coral fragments present close to the shoreline. Group C represents foraminifera assemblages that are distributed in shallow waters (6–7 m depth) in west Pulau Tioman. This group has a relatively lower quantity of stress-tolerant taxa (< 5%), but a relatively higher quantity of heterotrophic species, such as Eponides, Discorbinella, and Textularia. The presence of abundant sources of food may have promoted the increase in the number of heterotrophic taxa but they are limited by the dominance of stress-tolerant taxa13. Finally, group D represents most of the stations located in east Pulau Tioman with an FI of 6–9, indicating good water conditions for reef growth and recovery. The reduced coastal development activities observed in east Pulau Tioman may have decreased the impact of sedimentation and allowed a greater diversity of live corals to thrive3.To understand the similarity of the foraminifera assemblages around Pulau Tioman, a Q-mode cluster analysis was conducted. The results indicated that the benthic foraminifera assemblages can be classified into four major groups  Fig. . Three oThis study indicates that the FI is an excellent low-cost monitoring tool that can aid in assessments of water quality surrounding coral reefs. This finding also implies that the FI can be used together with the coral reef health index to determine the conditions and status of coral reefs in the region. Similarly, these indices can be applied to other coral reef ecosystems around Malaysia to determine their health. The range of FI values reported here shows that most of the sampling sites around Pulau Tioman are conducive to coral reef growth and recovery after exposure to any future bleaching events or temporary damages to the ecosystem. Several sites  with reduced FI values may not provide optimum conditions for reef recovery. Hence, marine park managers must closely monitor these touristic sites to prevent further coral reef deterioration. The carrying capacity of this ecosystem with respect to visitors and tourists at the Tioman Marine Park should be reviewed to determine optimum conditions for coral reef health, as indicated by the FI results.36. The island is influenced by two monsoonal systems: the northeast (November–March) and southwest monsoons (April–August)37. The maximum wave height during the northeast monsoon is ~ 4 m while during the southwest monsoon the height is < 1 m39. Pulau Tioman is surrounded by numerous coral reefs, with approximately 57–59 genera of hard corals distributed around the island3. Acropora, Montipora, and Porites are among the most common coral genera present around Pulau Tioman27. The extensive reef ecosystem surrounding Pulau Tioman is among the reasons why this island has been established as a National Marine Park by the Malaysian government40. The beauty of the island has attracted tourism-related activities since 199041. The sheltered west coast of Pulau Tioman  method48. Prior to heating, 5 g of each sediment sample was weighed. The samples were gradually heated until they reached 500–550 °C, followed by ignition for 4 h, cooled to room temperature (30 °C), and the final weight was recorded. The percentages of organic matter were calculated based on the formula provided in Heiri et al.49.The grain size analysis was conducted based on the dry-sieving method by Folk50. The Principal Component Analysis (PCA) was performed to investigate the relationships between the FI values and the sediment and water quality around Pulau Tioman. In addition, to investigate the relationships between the foraminifera species and the environment, canonical correspondence analysis (CCA) with a Monte Carlo permutation test was performed51. Foraminifera species with a relative abundance of > 10% in at least one sample were selected for the CCA and permutation test.To avoid using reworked samples, only foraminiferal species with a relative abundance of more than 2% were maintained for further statistical analysis. Indices, such as Fisher’s alpha diversity index (α), Shannon–Wiener species diversity (H′), and species evenness of Pielou (J′), were analyzed using the PAST  software version 3. Cluster analysis was performed to simplify the large datasets for easy recognition11 and Carnahan et al.29. The calculation of the FI was based on the equation proposed by Hallock et al.11. The values obtained from the calculation were interpreted based on Table The FI was used to determine the health status of the reef at Pulau Tioman. The foraminifera species collected around Pulau Tioman were identified and segregated into functional groups Table , as suggSupplementary Information.


Influenza A viruses cause a mild-to-severe respiratory disease that affects millions of people each year. One of the many determinants of disease outcome is the innate immune response to the viral infection. While antiviral responses are essential for viral clearance, excessive innate immune activation promotes lung damage and disease. The influenza A virus RNA polymerase is one of viral proteins that affect innate immune activation during infection, but the mechanisms behind this activity are not well understood. In this review, we discuss how the viral RNA polymerase can both activate and suppress innate immune responses by either producing immunostimulatory RNA species or directly targeting the components of the innate immune signalling pathway, respectively. Furthermore, we provide a comprehensive overview of the polymerase residues, and their mutations, associated with changes in innate immune activation, and discuss their putative effects on polymerase function based on recent advances in our understanding of the influenza A virus RNA polymerase structure.The online version contains supplementary material available at 10.1007/s00018-021-03957-w. A 100 years after the devastating influenza pandemic of 1918, influenza A viruses continue to pose a serious threat to human health. They infect up to twenty percent of the global population each year, resulting in several hundred thousand deaths –4.Many factors contribute to influenza disease severity, including underlying risk factors, bacterial co-infections, and the innate immune response to infection , 6. DuriActivation of the innate immune response is triggered by the replication of the viral genome . The infSeveral viral proteins, such as NS1, PA-X and PB1-F2, have well-established functions as innate immune modulators , 26. HowIn the last decade, significant advances have been made in understanding influenza A virus genetics, vRNA and vRNP structure, and the catalytic activity of the influenza A virus RNA polymerase . HoweverThe viral RNA polymerase transcribes and replicates the vRNA segments in the context of vRNPs Fig. A. WithinThe influenza virus RNA polymerase is comprised of three subunits: PB1, PB2 and PA  intermediate using a primer-independent initiation mechanism Fig. D. As theBesides Pol II and ANP32A, viral transcription and replication are assisted by a number of other cellular proteins, many of which also directly interact with the viral polymerase (reviewed in ). These Pathogen receptors, or pattern recognition receptors (PRRs), start the innate immune response during an infection. PRRs function by binding to conserved structures, or pathogen-associated molecular patterns (PAMPs), and activating signalling cascades that trigger expression of innate immune genes, such as type I and type III interferons. There are at least three PRR protein families involved in the recognition of an influenza virus infection, including toll-like receptors (TLRs), the nucleotide oligomerization domain (NOD)-like receptors (NLRs) and RIG-I-like receptors (RLRs) . In addiIn most cell types, the RIG-I signalling pathway Fig.  plays a Our understanding of the mechanisms behind the interactions between RIG-I and the viral promoter are complicated by the fact that the viral RNA polymerase shields the partially double-stranded promoter from RIG-I recognition in the context of an RNP. The RNA polymerase binds the first 10 residues of the 5′ terminus in a hook structure within a binding pocket consisting of PA and PB1 residues. The 3′ terminus, on the other hand, exists in at least three positions: either bound above the active site (A-site), on the outside of the PB2-N1 and PB1 thumb subdomain (B-site), or in the active site , an activator of PKR and RIG-I , 80. In The PB1 subunit of the RNA polymerase was recently found to inhibit RIG-I signalling by inducing autophagic degradation of MAVS . SpecifiThe PB2 subunit of the RNA polymerase also inhibits innate immune signalling by targeting MAVS , 84. TheAnother mechanism through which PB2 modulates MAVS-mediated signalling is by targeting TNF receptor-associated factor 3 (TRAF3), the adaptor protein of MAVS that is required for optimal signal transduction . TRAF3 iThe PA subunit of the viral RNA polymerase also inhibits innate immune signalling by binding to IRF3. This interaction prevents phosphorylation and nuclear translocation of IRF3, both of which are central for IFN expression . The samMitochondria serve as platform for the interaction between RIG-I and MAVS and thus play a prominent role in innate immune signalling , 93, 94.The mitochondrial targeting signal of PB2 has been mapped to PB2 residues L7 and L10, or N9 , 95. It Besides full-length replication products, the influenza virus RNA polymerase also generates aberrant replication products, including DVGs, mvRNAs, and svRNAs Fig. A 24, 9898–100. svRNAs, DVGs and mvRNAs all contain the conserved RNA promoter structure with 5′ triphosphate that forms a ‘panhandle’ in solution . In vitrThe molecular mechanisms underlying DVG and mvRNA formation are currently poorly defined. In one model, the internal deletions are generated when the viral RNA polymerase pauses during elongation, back-tracks to separate template and nascent strand, and finally translocates to a downstream template sequence to realign the nascent strand and continue nascent RNA extension Fig. B 22, 9898. Such Several viral factors have been associated with the formation of aberrant RNAs. Recent studies have shown that elongation defects can be induced experimentally by limiting the availability of NP, an important elongation factor and key component of RNPs, suggesting that impaired elongation or aberrant encapsidation play a role in DVG or mvRNA formation , 111. InWe can learn more about the potential mechanisms underlying aberrant RNA generation and their role in innate immune activation by studying the RNA polymerase mutations that affect their formation. High levels of mvRNA production by the polymerases of highly pathogenic avian H5N1 and 1918 pandemic viruses are in part determined by avian-adaptive mutations in the PB2 polymerase subunit of those strains . IntroduSynthesis of aberrant RNAs is also affected by the fidelity of the viral polymerase. A V43I mutation in the PB1 subunit, which reduces the mutation rate of RNA synthesis by approximately twofold in some genetic backgrounds, was also shown to lower levels of mvRNA synthesis by the polymerases of the 1918 pandemic and H5N1 strains , 115. InSimilar to mvRNA synthesis, DVG formation is affected by mutations in the RNA polymerase. Two of these mutations, PB2 A221T and PA D529N, were identified in a virus isolated from a fatal case of pdm09 (H1N1) influenza . InteresAberrant polymerase activity can also be the result of defects in viral transcription. Influenza virus transcription is dependent on cap-snatching and the binding of the C-terminal domain of PA to a Ser5 phosphorylated CTD of Pol II. Mutations in key PA residues involved in this interaction, K635A and R638A Fig. F, not onThe influenza A virus RNA polymerase plays a major role in the adaptation of avian influenza virus strains to mammalian cells. Several major host-adaptive mutations induce a strong activation of innate immune responses which correlates with the improvement in viral replication.One of the most well-studied polymerase adaptations is a E-to-K mutation at residue 627 of PB2 . This muRecent experiments have shown that a lysine at position 627 is essential for a stable interaction of the viral RNA polymerase with mammalian host-cell protein ANP32A . ANP32A In some avian influenza virus strains, a Q591K mutation in the PB2 627 domain [G590S and Q591R in pdm09 (H1N1)] can compensate for the absence of the E627K signature and support the activity of avian-adapted polymerases in mammalian cells , 127. LiPB2 mutation D701N has also been associated with improved replication and higher levels of innate immune activation , 128. HoTo explore the distribution of published polymerase mutations that affect innate immune responses and identify potentially novel clusters of interest, we mapped those mutations to the polymerase subunits Fig. . In partThe PB2 Mid-link domain might play a key role in stabilizing the conformational rearrangements of the RNA polymerase as it transitions from one state to another or in encapsidation of the nascent RNA strand. It forms extensive, transient interactions with the flexible domains of PA and PB2 in various conformations of the viral polymerase. In addition, analysis of the ANP32A-supported replicase suggests that the Mid-link domain may play a role in nascent strand egress or encapsidation. In the dimer, residues 252–273 and 519–523  of the replicating polymerase face the encapsidating polymerase but are not directly interacting with it. Instead, these residues face a groove that separates the two polymerases in the dimer and which the emerging 5′ terminus of the nascent strand must bridge to reach the promoter binding pocket of the encapsidating polymerase  virus, which contained a dysfunctional NS1 of the bat influenza A virus . The mutFlaviviridae, Picornaviridae and Coronaviridae families are also known to specifically target and inhibit innate immune signalling [The influenza A virus RNA polymerase plays multiple roles in the innate immune response to influenza virus infection. Not only can the subunits of the RNA polymerase affect innate immune activation by interfering with the components of the cellular signalling pathways, the enzyme can also produce immunostimulatory RNA species. These activities of the RNA polymerase are not unique to influenza A viruses, as RNA polymerases of viruses belonging to gnalling –139, whignalling .Despite recent advances in our understanding of the structure and the immunostimulatory and immunoinhibitory activities of the influenza A virus polymerase, many fundamental questions about the molecular mechanisms involved remain unanswered. These include, but are not limited to, (i) how does RIG-I gain access to viral RNA  in the context of a fully assembled RNP; (ii) what is the molecular mechanism underlying the generation of DVGs and mvRNAs; (iii) why do aberrant viral RNAs trigger innate immune responses more efficiently than full-length viral RNA segments; (iv) does aberrant RNA synthesis confer any evolutionary advantage, and (v) are the immunosuppressive and enzymatic activities of the viral polymerase separated in space and time? To find answers to those questions, existing and novel RNA polymerase mutants can be used. Screening approaches in combination with next generation sequencing have proven to be particularly powerful for the identification of such immunostimulatory RNA polymerase mutants , 141. HoThe immunomodulatory or -stimulatory activity of the viral polymerase could also guide the development of novel antiviral treatments. Even though current drugs targeting the influenza virus polymerase primarily focus on blocking its activity, targeting its immunomodulatory function could potentially have an added benefit of activating the host’s natural immune defence during infection. However, care must be taken to not over-stimulate the innate immune response. Additionally, DVG-containing influenza viruses or cloned DVGs have been proposed as both influenza-specific and a broad-spectrum antiviral treatment due to their interfering and immunostimulatory activity , 142.The knowledge of the processes by which influenza virus polymerase stimulates or inhibits innate immune responses can also be used in rational vaccine design. Several PB1, PB2 and NP mutations in the current live-attenuated influenza vaccine confer its cold-adapted, attenuated and temperature-sensitive phenotype , while aSupplementary file 1 (DOCX 210 KB)Below is the link to the electronic supplementary material.


Secondary metabolites (SMs) play crucial roles in the vital functioning of plants such as growth, development, defense, and survival via their transportation and accumulation at the required site. However, unlike primary metabolites, the transport mechanisms of SMs are not yet well explored. There exists a huge gap between the abundant presence of SM transporters, their identification, and functional characterization. A better understanding of plant SM transporters will surely be a step forward to fulfill the steeply increasing demand for bioactive compounds for the formulation of herbal medicines. Thus, the engineering of transporters by modulating their expression is emerging as the most viable option to achieve the long-term goal of systemic metabolic engineering for enhanced metabolite production at minimum cost. In this review article, we are updating the understanding of recent advancements in the field of plant SM transporters, particularly those discovered in the past two decades. Herein, we provide notable insights about various types of fully or partially characterized transporters from the ABC, MATE, PUP, and NPF families including their diverse functionalities, structural information, potential approaches for their identification and characterization, several regulatory parameters, and their modulation. A novel perspective to the concept of “Transporter Engineering” has also been unveiled by highlighting its potential applications particularly in plant stress (biotic and abiotic) tolerance, SM accumulation, and removal of anti-nutritional compounds, which will be of great value for the crop improvement program. The present study creates a roadmap for easy identification and a better understanding of various transporters, which can be utilized as suitable targets for transporter engineering in future research. Arabidopsis thaliana genome encodes transporter proteins), only a handful are having their function and substrate specificity well deciphered by experimental evidence, especially the ones involved in the transport of SMs. Thus, it is challenging to find the appropriate candidates for transporter engineering to accomplish the long-term goal of systemic metabolic engineering for the betterment of plants as well as for medicinal benefits.The transport of metabolites in the plants is an indispensable phenomenon for the dynamic functioning of numerous cellular processes ranging from growth, development, survival, defense, maintaining homeostasis, and coordination in the system . SpecifiConsidering this, better knowledge of SM transporters, particularly their diversity, functionality, regulatory parameters, approaches for their identification, and characterization is a pre-requisite for future research. In this review article, we attempt to provide a perspective and create a foundation in the field of systemic metabolic engineering by utilizing the most feasible approach of engineering the potential transporters by modulating their expression. Here, we are discussing the existing data as well as updating the understanding of plant SM transporters. Few of the previously published reviews  have mosNicotiana tabacum, the transport of nicotine from roots to leaves is mediated by the xylem pathway  cells  cells  has emer2) cells . Interes2) cells .The present review provides the current perspective of a large number of SM transporters particularly discovered in the past two decades from various important plant species . Herein,Solanum lycopersicum  (Lotus japonicus (Hevea brasiliensis)  . Besidesinifera) , pineappinifera) , Lotus japonicus , and rubliensis)  have alsliensis) . A typicliensis)  and if tliensis)  amongst liensis) . Althougliensis) . The altliensis) . The cryliensis) .Coptis japonica  namely berberine is produced, which is known for its antimicrobial properties. Berberine is synthesized in the roots and possibly further accumulates into the rhizome of the plant. Two transporters from the ABCB subfamily i.e., CjABCB1 and CjABCB2 have been characterized and found to be involved in the influx of berberine into the rhizome  transporters. PDR is part of the ABCG subfamily that particularly has an arrangement of NBDs and TMDs in reverse orientation  from the plant. General assumption dictates that VOCs may simply emit out from their biosynthetic site such as flowers via the process of diffusion; but being a slow process, diffusion can create the temporary accumulation of VOCs at the membranes, which might be toxic for the integrity and functioning of the cellular membranes. Thus, active transport has also been predicted for the emission of VOCs. To identify the putative VOC transporter,  hybrida .Crocus sativus (saffron), which is one of the most expensive and valuable spices in the world. The red appearance of saffron arises due to the presence of glycosylated apocarotenoids commonly known as crocins, which are probably synthesized in the cytosol and further transported to vacuolar lumen for storage. Since saffron is obtained from the dried stigma of C. sativus, its transcriptome was searched for putative transporters. The analysis identified CsABCC4a protein which is expressing specifically in the stigma and might be employed for the transport of crocins to the vacuole. Further, the co-expression studies of CsABCC4a with crocin biosynthetic pathway enzymes also showed a positive correlation. Finally, the localization and functional studies have validated the potential role of CsABCC4a in the vacuolar accumulation of crocins  ABC transporters (CjABCB1/2) which suggested that berberine is synthesized in the roots and further transported to the sink tissue rhizome  , which is a highly valuable widely grown commercial crop. This variety naturally produces brown fibers, thus it is also named brown cotton, and it has been reported that PA is the major substance behind its brown coloration. The naturally occurring brown color benefits the textile industry by reducing the dyeing and processing cost  and anthocyanins are also mediated by MATE transporters. M1, AM3)  and putaM1, AM3) , which aocyanins . Howeverocyanins . Anothering cost . Based o studies . In addi studies . Similarhirsutum , which mA. thaliana and reported to be involved in the transport of adenine, cytosine, and purine derivatives . Further, the substrate specificity of AtPUP1 was determined by functional complementation assays in a yeast mutant, which has shown that AtPUP1 is a high-affinity transporter for adenine and cytosine  , which mediate the transport of various opiates belonging to a diverse class of BIA. P. somniferum is the prime natural source of BIAs and several therapeutic compounds or their precursors such as morphine, codeine, and thebaine  1–6, due to their clustering with BIA biosynthetic pathway genes  . In toba-1 (NUP) . Therefoot cells  and probot cells . Similarot cells  has alsothebaine . The genay genes . The opiay genes .O. sativa, Z. mays, Sorghum bicolor, S. lycopersicum, Carica papaya, L. japonicus, etc. The most studied plant, A. thaliana was found to have 50 NPFs, followed by 20 and 21 in Physcomitrella patens and Marchantia polymorpha, respectively. On the other hand, a very high number has been reported in M. truncatula and G. max i.e., 92 and 114, respectively , which is known to be a native vegetable of china which has high nutritional value along with anti-carcinogenic and antioxidant properties due to the presence of various glucosinolates, carotenoids, and phenolic compounds  and MIAs. Glucosinolates are a specific class of plant SMs derived from amino acids and many of the GSLs have high nutritional value due to their rich nitrogen and sulfur content . GSLs arthaliana  are repothaliana . The funompounds . In B. oand buds  with a vlidation . Similarand GTR2 . The ind unclear .C. roseus that specifically transports a metabolite strictosidine which belongs to the class of MIA. Strictosidine is an intermediate metabolite in the MIA biosynthetic pathway and it has been hypothesized from transcriptomic data of C. roseus that CrNPF2.9 transporter is co-regulated with early MIA pathway genes, which perhaps, hints toward its involvement in the transport of MIA pathway intermediates  treatment  and bacterial (Pseudomonas syringae) pathogens via JA signaling pathway  has been well established in the enhanced production of several SMs including alkaloids, polyphenols, and terpenoids  which pris genes . On the reatment . Similarhe plant . In addihe plant . One suche plant . As reviectively . As a rereatment . Whereasethylene . Moreoveerbivory . Likewis pathway . The Sololerance . Similarc manner .cis-regulatory elements (CRE) which are responsive toward various regulatory factors such as hormones, environmental conditions, biotic and abiotic stresses can control the expression of their respective genes at transcriptional levels. The common CREs have been identified in the promoter region of various potential ABC transporter genes of Capsicum and Brassica species  , pathogens (WRKY71OS), and wounding (WBOXNTERF3)  into the vacuole and further massive accumulation of E3′G in hairy roots confirms TF induced uptake by MtMATE1 transporter  with potential MATE transporters of tomato (V. vinifera was found to be positively regulated with the increase in expression of TFs namely VvMYBPA1 and VvMYBPA2 (A. annua (M. truncatula (C. roseus (The presence of  species . CaABCC ATCOR15) . While, XNTERF3) . Additioive CREs . Likewisf NpPDR1 . Similarnsporter . Similarocyanins . Another SlMTP77 . Additiof tomato . FurtherVvMYBPA2 . SeveralA. annua , TSAR1/Tuncatula , and ORC. roseus  have bee. roseus .A. thaliana are unveiled by its comparative study with a functionally characterized mammalian ABCC7 transporter which ultimately deciphered a conserved mechanism of regulation i.e., protein phosphorylation at serine residues by PINOID kinase. The addition of phosphate group occurs at S634 residue on the linker regulatory domain, which joins the NBD folds of the ABCB1 and such chemical modification is reported to modulate the function of the transporter. Besides, its negative regulation has also been observed when phosphorylation occurs at a different non-linker location  such as phosphorylation, ubiquitination, acetylation, methylation, and glycosylation, etc. The role of phosphorylation and ubiquitination have been studied in various plant transporter proteins including potassium/nitrate/ammonium transporters and inorganic phosphate transporter , respectively . In contlocation . Another1 kinase . Additio1 kinase . Various1 kinase . As repo1 kinase . Furtherubstrate , whereasubstrate .Allium genus  and Brassica juncea (BjGTR2) was achieved by inserting early stop codons in their genes via mutagenesis and this facilitated a more than 60% reduction in the accumulation of seed glucosinolates   . As prevphyllene .Metabolic engineering of plants has been given a lot of importance in recent years due to its immense potentials. Various strategies have been deciphered to increase the yield of SMs and their accumulation. In the majority of earlier research efforts, the increase in metabolite yield was achieved through the modulation of the biosynthetic pathway genes. However, advancement of knowledge in the area opens possibilities of regulation of potential transporter genes for precise metabolic engineering. The transporters of plant SMs are the key players that have an impact not just at the site of metabolite synthesis and accumulation but systemically affect the plant metabolic system in one way or the other. In the recent past, several SM transporters have been identified in plants but their functional validation and substrate specificity determination is a huge challenge and equally critical for the metabolic engineering of plants. The present review emphasizes the knowledge gain in the area concerning SM transporters and their modulation. The transporter engineering, particularly for plant stress (biotic and abiotic) tolerance, SM accumulation, and removal of anti-nutritional compounds will be of great future potential. Further, the leverage of this technology can be extended to many medicinal, agricultural, aromatic, and other economically important plants for their optimum utilization and value addition. However, for efficient transporter engineering, more emphasis is required to identify the possible involvement of multiple transporters and/or their cross-talks for the long-distance mobilization of SMs. In addition, more and more transporters associated with secretion and storage should be annotated to get better insight into their functionality and possible applications. Similarly, the workings of the two-way transport system and the source-sink relationship linked to the distribution of SM pool need to be investigated in detail. Overall, it is realized that transport engineering is a dynamic and fairly complex process and emerges as a critical tool for systemic metabolic engineering in plants. However, it is also equally important to address the associated challenges using modern experimental tools and approaches to make it more effective and harness its fullest potential.PN wrote the original draft. PP conceptualized the idea, supervised, and critically reviewed the manuscript. Both authors significantly contributed to finalizing the manuscript and approved it for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dimethylsulfoniopropionate (DMSP) is a key organic sulfur compound that is produced by many phytoplankton and macrophytes and is ubiquitous in marine environments. Following its release into the water column, DMSP is primarily metabolised by heterotrophic bacterioplankton, but recent evidence indicates that non-DMSP producing phytoplankton can also assimilate DMSP from the surrounding environment. In this study, we examined the uptake of DMSP by communities of bacteria and phytoplankton within the waters of the Great Barrier Reef (GBR), Australia. We incubated natural GBR seawater with DMSP and quantified the uptake of DMSP by different fractions of the microbial community . We also evaluated how microbial community composition and the abundances of DMSP degrading genes are influenced by elevated dissolved DMSP levels. Our results showed uptake and accumulation of DMSP in all size fractions of the microbial community, with the largest fraction (>8 µm) forming the dominant sink, increasing in particulate DMSP by 44–115% upon DMSP enrichment. Longer-term incubations showed however, that DMSP retention was short lived (<24 h) and microbial responses to DMSP enrichment differed depending on the community carbon and sulfur demand. The response of the microbial communities from inside the reef indicated a preference towards cleaving DMSP into the climatically active aerosol dimethyl sulfide (DMS), whereas communities from the outer reef were sulfur and carbon limited, resulting in more DMSP being utilised by the cells. Our results show that DMSP uptake is shared across members of the microbial community, highlighting larger phytoplankton taxa as potentially relevant DMSP reservoirs and provide new information on sulfur cycling as a function of community metabolism in deeper, oligotrophic GBR waters. Phytoplgellates ,3,4,5. Igellates . Likewisgellates . Severalgellates ,9,10, whgellates ,12,13.Intracellular or particulate DMSP (DMSPp) is transported from the synthesising cell into the external seawater environment via exudation, viral lysis or cell senescence ,15,16. OSeveral genes have been identified as key DMSP degraders in the marine environment. The gene DmdA, is the first cataboliser in the demethylation pathway and is the most abundant DMSP degrading gene in the oceans, estimated to be present in 37–58% of all bacterioplankton ,29. In cProchlorococcus, Synechococcus, as well as some eukaryotic phytoplankton from the dinoflagellates, cryptophytes and diatoms groups have been shown to take up dissolved DMSP [In addition to bacterioplankton, very low or non-DMSP producing phytoplankton have been shown to play a role in DMSP cycling. Prokaryotic phytoplankton from the groups ved DMSP ,34,35,36ved DMSP , the utived DMSP . Identifved DMSP ,26,39, wved DMSP ,41.−1 [In this study, we examined the uptake of DMSP by natural microbial communities of the Coral Sea surrounding the Great Barrier Reef (GBR) in Australia. These waters are characterised by high solar radiation, low nutrient concentrations and often dominated by cyanobacteria ,43,44,45−1 ). MovingThis study was conducted during an oceanographic voyage on the RV Investigator in October 2016. Two sites within the Great Barrier Reef were chosen: a southern open ocean (depth ~400 m) site   and a shallower (~45 m) northern inner-reef site , located between the coast and the reefs . The inner reef site was located ~13 km from the closest reefs of John Brewer Reef, Rib Reef and Fore and Aft Reefs; the deeper outer reef was located ~25 km from Bills Reef . At bothAt each site, two separate incubation experiments were conducted: (1) a short-term study (7 h) to quantify DMSP uptake by different size fractions of the microbial community, and (2) a longer (120 h) experiment to investigate the influence of DMSP on community structure and DMSP degradation gene regulation.2SO4  and kept at −80 °C until processing [To study the uptake of DMSP by the microbial community of the Great Barrier Reef, 28 L of seawater was collected and transferred to 4 L-polycarbonate bottles with no headspace. Bottles were separated into three treatments, in triplicate: controls (unamended seawater), DMSP addition (+10 nM DMSP) and fixed (dead). The fixed sample was incubated with glutaraldehyde  for 1 h, before DMSP  was added, to control for any passive uptake of DMSP by the cells. Following DMSP addition, the bottles were closed with screw caps and inverted gently to dissolve the added compound. All flasks were then incubated for up to 7 h in an on-deck flow-through incubator. At four time points  each bottle was subsampled in triplicate for quantification of DMSP total and dissolved  concentrations, either as a whole water sample or a gravity filtered  sample (3 mL), respectively. Sub-samples were acidified with Hocessing . After 7To study microbial community changes caused by DMSP enrichment, 30 L of water were divided across 15 × 2 L polycarbonate bottles (no headspace). Bottles were separated into two treatments: six controls (unamended) and nine amended with DMSP . Bottles were then incubated in an on-deck flow-through incubator until sampling at three time points . At each time point, subsamples (3 mL) for DMSP total and dissolved  were taken and acidified as described above.2 and subsequently eluted onto a capillary column . For experiment 1, particulate DMSP (DMSPp) was measured directly on the filtered size fractions, where filters were placed into vials with NaOH and quantified as total DMS as described above. Particulate DMSP for experiment 2 was calculated by subtracting DMSPd from DMSPt. Integrated peak areas for each sample were quantified against a calibration curve obtained from solutions prepared with known amounts of DMSP hydrolysed with NaOH and analysed using the same method as the samples. For determination of DMSP lyase activity (DLA) rates of the starting communities, subsamples (600 mL) were filtered first through a GF/C filter  for the phytoplankton fraction rates (DLAp), and then 300 mL of the phytoplankton-free sample filtrate was filtered onto a 0.22 μm polycarbonate filter for bacterial DLA (DLAb). Both filters were placed into cryotubes, flash frozen with in liquid nitrogen and stored in −80 °C until analysis. DMSP lyase activity was determined via direct injection of 500 µL of headspace  as previously described [Biogenic sulfur compounds  were quantified as total DMS after conversion with 100 mg of NaOH and measured using a gas chromatograph  coupled with a flame photometric detector (FPD). Samples were purged with He (60 mL/min for 4 min) while cryo-trapped in liquid Nescribed .Prochlorococcus, Synechococcus and picoeukaryotes were discriminated using side scatter (SSC) and red and orange fluorescence [Quantification of microbial populations via flow cytometry was performed using a Beckman Coulter Inc flow cytometer . Populations of rescence . Samplesrescence ,54. DataNutrients, measured in the initial sample  and at T24, T72 and T120 h from the incubations were carried out on board as previously described . Data weIn both incubation experiments, the remaining sample (~1.9 L) at the final time-point was filtered through a 0.22 µm polycarbonate filter, which was subsequently snapped frozen and kept at −80 °C. DNA was extracted using the Powerwater DNA isolation kit  following manufacturer’s instructions, and the DNA concentration was quantified using a Nanodrop spectrophotometer . Extracted DNA was stored at −20 °C until analysis. Prior to analysis the DNA was split into two fractions, with one used for the analysis of prokaryotic and eukaryotic microbial diversity using (16S and 18S rRNA) amplicon sequencing, and the second fraction used for the quantification of DMSP degrading genes using real time quantitative Polymerase Chain Reaction (qPCR) described below.For 16S amplicon sequencing, 20 µL aliquots of ≥10 ng/µL DNA were used and amplified using the 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) primers and subsequently sequenced using the Illumina MiSeq platform   following the manufacturer’s guidelines. The V4 Primers  (Nextera-tagged primers) were used for 18S rRNA, with a thermocycling profile starting at 94 °C for 3 min; then 30 cycles of: 30 s at 94 °C, 60 s at 57 °C, 90 s at 72 °C; and a final 10 min at 72 °C. Amplicons were subsequently sequenced using the Illumina MiSeq platform  .https://zenodo.org/record/3362765#.YL2O9y0RrUI, accessed on 19 April 2021). The dataset was further cleaned by removing sequence variants (ASVs) identified as chloroplasts, mitochondria and ASVs (abundance cut-off of 0.03%). Sequences were then rarefied to the same depth to remove the effect of sampling effort upon analysis. Raw FASTQ data files for the 16S rRNA and 18S rRNA assay were deposited in NCBI Sequence Read Archive (SRA) under Bioproject number PRJNA755150.Gene sequencing reads were processed to an abundance table of Amplicon Sequence Variants (ASVs) from demultiplexed paired fastq files using DaDa2 (version 1.14.1 ). Briefl1 copies L−1 and 106 copies L−1 of prepared cloned gene fragments from environmental samples. Working stock solutions of a concentration of 1010 copies L−1 of each gene were prepared from purified gene fragments using a Qubit fluorometer . All DMSP degradation genes were normalised to 16S gene copy number  and DMSP cleavage (DddP) were enumerated using quantitative polymerase chain reaction  with the Biorad CFX384 manager software . Samples were run in technical triplicate of total reaction volume 5 µL containing; 2.5 µL of 2 × Sensifast Hi-rox master mix , 0.2 µL of each forward and reverse primer and 2.1 µL of DNA template). Thermocycling conditions of DmdA ,  and Ddd  involved a 5 min denaturation step at 95 °C, followed by 40 annealing temperature cycles of 54 °C, 42 °C and 41 °C, respectively  or site and treatment (DMPSp), as fixed factors. A Tukey’s post hoc test was performed to locate differences and account for multiple comparisons. Using a resemblance matrix based on Euclidean distance, a two factor Permutational Multivariate Analysis of Variance (PERMANOVA) with pair-wise comparisons was used to determine significant changes or differences in DMSPt and DMSPd concentrations over time and between treatments and to analyse the differences between treatments over time for the relative abundance of dmdA/A1, dmdA/Dall and DddP genes data. Differences in bacterioplankton (16S) and phytoplankton (18S) community composition between treatments over time (exp. 2) were analysed using PRIMER v6 statistical package . Data weX) consistently low (≤0.02 µM) at both sites and for both incubation experiments. Phosphate concentrations were similar at both sites, ranging from 0.038 to 0.050 µM in the inner reef, and 0.049 to 0.066 µM in the outer reef, while silicate concentrations were 0.798 and 0.882 µM in the outer reef compared with 0.500 and 0.623 µM at the inner reef site for the two incubation experiments  of Synechococcus inside the reef compared with outside the reef (differing by an order of magnitude), while Prochlorococcus abundances were highest for experiment 1  at the inner reef site (6.56 ± 2.7 × 105 cell mL−1) and experiment 2 (5.59 ± 0.53 × 105 cell mL−1) at the outer reef site. Heterotrophic bacterial abundances were higher at the inner reef site (0.79 ± 0.16 × 105 cell mL−1) compared to the outer reef site (0.30 ± 0.05 × 105 cell mL−1), with differences in abundance between experiments for the inner reef site  and Picoeukaryote abundance was more than double inside (1490 ± 250 cell mL−1) than outside the reef .Picoplankton abundances varied between sites and experiments, with generally higher abundances at the inner reef site . There wp = 0.001) and outer reef  incubations (p = 0.001), representing a loss of initial DMSPt of ~43% in both treatments  and outer  reef sites over time  in DMSPp in the largest (>8 µm) fraction (p = 0.0026), but with no effect of DMSP enrichment. In the 3–8 µm fraction, DMSPp levels were higher in the inner reef , but enrichment did not result in a further increase in DMSPp. For the largest fraction (>8 µm), the inner reef had higher levels of DMSPp, and enrichment increased DMSPp at both sites . There was no change in DMSPp in fixed cells with DMSP enrichment, which remained low in all fractions (Particulate DMSP levels varied according to size fraction and treatment C. Withinfraction C. For thractions C.−1) than those of the bacterial (DLAb) fraction  were three times higher than DLAp rates  in the bacterial population of the inner reef compared with the outer reef site  than the outer reef site  differed between sites . The inneef site C. The coProchlorococcus, while the other important photosynthetic cyanobacterium Synechococcus was present at 3.6% in the inner reef site, yet <1% at the outer reef. Phytoplankton community composition was similar between sites  and monitored changes in microbial communities and sulfur compounds over five days. Macronutrient concentrations over time differed between the inner and outer reef experiments, where the inner reef, phosphate dropped below detection limit by 120 h, and silicate concentrations increased from initial concentrations . For thep = 0.001) from 17.36 to 10.64 nM in the control and 25.72 to 13.54 nM in the DMSP-enriched incubations  disappeared after 24 h. Outside the reef, DMSPt declined  from 17.35 to 7.08 nM in the control and from 33.38 to 6.9 nM in the DMSP-enriched samples (p = 0.005) disappearing after 24 h, but at a higher loss rate, 9.36 nM day−1 for the controls and 19.92 nM day−1 for the DMSP-enriched samples.For the inner reef site, DMSPt declined significantly , followed by a steady, slow continuous decrease over the remaining 96 h  from 9.33 to 2.50 in the controls and from 22.91 to 1.94 nM in the DMSP-enriched samples (−1 (controls) and 18.56 nM day−1 (+DMSP). This loss of DMSPd over 120 h equates to a loss of the initial DMSPd of approximately 73% in the controls and ~91% in DMSP-enriched bottles. As with DMSPt, the rapid decline meant that initial differences in DMSPd between treatments for the inner  and outer  reefs disappeared after only 24 h.For the inner reef, concentrations of DMSPd remained constant at ~2 nM in the control over time, while DMSP-enriched samples showed a rapid and significant decline in the first 24 h  with a loss rate of 1.07 nM day−1. Conversely, DMSPp remained constant at ~6 nM for the controls yet significantly diminished from 10.46 nM to 4.94 nM for the DMSP-amended samples  at a rate of 1.10 nM day−1 in the outer reef samples , DddP  and DmdA/A1  increased over the 120 h, with faster rates of increase  in the control compared with DMSP-enriched samples (p = 0.001) and DmdA/A1  decreased significantly over 120 h (p = 0.003) and maintained a higher relative abundance  than the controls (−1) for the inner reef site. In contrast, DLAp rates were approximately four times higher than rates of DLAb for the outer reef site , and were an order of magnitude lower than those measured for the inner reef community (data not shown).Contrasting trends in DMSP degradation gene abundance were evident between the two sites, with significant increases over time for the inner reef community, compared with a stable or declining gene abundance over time for the outer reef community D–F. Insi samples D–F. The er 120 h E,F. Despcontrols D. DMSP lProchlorococcus 34% and Synechococcus 10%) and SAR11 clade (20%), together making up 64% of the community (p = 0.002), with Rhodobacterales, Synechococcales, Actinomarinales, SAR11 clade and Rhodospirillales forming the major groups contributing to 54.6% of the cumulative difference (Sulfitobacter), which reached 50% in the controls, compared with only 8% in the DMSP-enriched samples . In both treatments we saw a decline in relative abundance of Synechococcales after 120 h , where final abundances were lower in the controls (<10%) compared with the +DMSP (~28%) treatment . For the outer reef, the major groups contributing to 53.61% of the cumulative difference were Rhodobacterales, Synechococcales, Actinomarinales and Sphingomonadales (p = 0.008). In the +DMSP treatment this shift towards Rhodobacterales, (predominantly Sulfitobacter) was retained for 120 h, becoming the dominant order (~42%) within the community at 120 h, whereas in the control, the relative abundance returned to low numbers (~6%) by the end of the experiment (p = 0.001), but remained higher  in the control (~27%) than the +DMSP (~7%) treatment  with five orders—Diatoms , Dino-Group , Gymnodiniales, Suessinales and Diatom —contributing to 51.41% of the dissimilarity between treatments (Phytoplankton community composition of the inner reef was dominated by dinoflagellates belonging to seven different taxonomic groupings . Dino-Greatments .p = 0.001), with Dino-Group , Diatoms , MAST-Group, Gymnodiniales and Stramenopiles as the major contributors to 53.49% of the dissimilarity between treatments  of diatoms . As witheatments .2 = 0.67; p = 0.0003) and DddP , but only for the inner reef site  showed a loss of DMSPt, for both sites and treatments, indicating that DMSP was escaping from the system. This could occur via bacterial conversion to DMS, DMSO and MeSH or by photochemical oxidation to DMSO ,71,72. AProchlorococcus, typical of warm oligotrophic areas, due to its adaptability to thrive in nutrient poor waters [Synechococcus sp. KORDI-100, has been found to have a DMSP lyase gene [Prochlorococcus and SAR11 were present in high relative abundances at both sites, the fact that DMSP showed higher accumulated in the small fraction of the outer reef, suggests that rather than differences in microbial community composition, it was the metabolic state of the community driving this difference, whereby the bacterial community of the outer reef site were preferentially taking up DMSPd to meet their sulfur and carbon demands for growth [At both reef sites, prokaryotic communities were dominated by the photosynthetic cyanobacteria r waters ,75. Becar waters ,76, it iase gene . Given tr growth ,27. Giver growth , on the a signature was twice as high at the inner reef site compared with that of the outer reef, indicating higher phytoplankton biomass in the inner reef surface waters. As such, we propose that the dinoflagellates accounted for the high proportion of DMSPp in the >8 µm fraction, but that the main taxa responsible for the uptake of additional DMSP in the +DMSP treatment be attributed to the diatoms, which have been shown to take up and accumulated DMSP in high concentrations [We found dinoflagellates dominated the reef waters from both sites . Given ttrations . The absAt both sites, the most abundant DMSP degrading pathway was demethylation, represented by the gene DmdA . Similar relative abundances of these DMSP degradation genes using qPCR have been reported for the North Pacific subtropical gyre . The Dmd−1) within previously reported values . The nM d−1; , in acco one day .Emiliania huxleyi [Phaeocystis spp. [To date, DMSP lyase activity (DLA) in phytoplankton has only been shown in  huxleyi , Phaeocytis spp.  and difftis spp. ,88, all tis spp. ,26,27. IAs with the short-term experiments, DmdA/Dall was the dominant DMSP degradation gene throughout the experiment (120 h) with similar relative abundances at both sites at the outset when most of the DMSP was lost from the system, suggesting that most of the DMSP was degraded through demethylation ,23. ThesSulfitobacter, a genus within the Rhodobacter order and the dominant genus in our study, have been previously linked with these genes and shown to use DMSP as their sole carbon source for MESH production [In favouring DMSP utilisers, enrichment with DMSP can disrupt competitive hierarchies in microbial communities, allowing some taxa to proliferate over others. We saw DMSP-related changes in the phytoplankton community over the 120 h at both sites, predominantly between the diatom and dinoflagellate groups, indicating an interplay between DMSP sources and sinks within the phytoplankton community. For the prokaryotic community we measured key changes over time, some of which were in response to DMSP enrichment and could be linked with changes in gene abundance . In partoduction . These oIn this study, we evaluated the uptake of DMSP by different fractions of marine microbial communities of the GBR and revealed that both bacteria and phytoplankton from natural reef waters can take up DMSP over short time scales. Specifically, DMSP enrichment revealed the main sink for DMSP comprised of taxa from the largest microbial fraction. By investigating the main DMSP catabolising genes over 120 h, we were able to show that despite similarities in microbial community composition, strong differences in DMSP catabolism and thus influence on sulfur cycling are possible. We found that taxa from the Rhodobacterales order best explained the increases in DMSP catabolising genes, but this relationship was only found for the inner reef site. The differences in DMSP lyase activity, catabolism and community structure observed between the two reef sites suggests that at the inner reef site sulfur and carbon demands were largely satisfied by existing DMSP availability, meaning that DMSP addition had minimal impact on the microbial community from these waters, with lyase activity dominating the conversion of DMSP to DMS. On the other hand, the absence of a strong response to DMSP enrichment for the outer reef bacterioplankton community , when taken in the context of the low DLA rates measured, suggests minimal DMSP cleavage to DMS occurring, and therefore we may presume that any DMSP in the system was being converted to MeSH or lost from the system via oxidation. Overall, our findings contribute to an improved understanding of the fate and function of DMSP in tropical marine ecosystems and identify microbial DMSP sinks and sources within the deeper, oligotrophic waters of the GBR.