Gammaherpesvirus infection triggers the formation of tRNA fragments from premature tRNAs

Abstract

Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. In addition, mounting evidence supports biological function for tRNA cleavage products, including in the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to enhanced tRNA transcription. However, whether this has any influence on tRNA transcript processing, viral replication, or the host response is not known. Here, we combined two new approaches, sequencing library preparation by Ordered Two Template Relay (OTTR) and tRNA bioinformatic analysis by tRAX, to quantitatively profile full-length tRNAs and tRNA fragment (tRF) identities during MHV68 infection. We find that MHV68 infection triggers both pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tRFs. OTTR-tRAX revealed not only host tRNAome changes, but also the expression patterns of virally-encoded tRNAs (virtRNAs) and virtRFs made from the MHV68 genome, including their base modification signatures. Because the transcript ends of several host tRFs matched tRNA splice junctions, we tested and confirmed the role of tRNA splicing factors TSEN2 and CLP1 in MHV68-induced tRF biogenesis. Further, we show that CLP1 kinase, and by extension tRNA splicing, is required for productive MHV68 infection. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing.

Figure 1.. Benefits of OTTR-seq for tRNA sequencing.

Traditional RNA-seq uses retroviral reverse transcriptases (RTs) with low processivity through tRNA modifications, resulting in depletion of modified transcripts in final sequencing libraries. TGIRT-seq uses the more processive TGIRT enzyme, but allows partial/incomplete cDNA products into library assembly where they are indistinguishable from biologically-relevant tRFs. The high processivity and template jumping activity of the OTTR RT used in OTTR-seq ensures that highly modified tRNAs and genuine tRFs (and not partial cDNA products) are sequenced.

Figure 2.. OTTR-seq robustly captures host tRNAs.

(A) Distribution of reads mapping to various small RNA classes using OTTR-seq (OTTR) or DM-tRNA-seq (DM) prepared from mock- and MHV68-infected MC57G mouse fibroblasts at an MOI=5 for 24 hours. (B) Normalized read counts for host tRNA isodecoders detected in mock-infected libraries using OTTR-seq. The dotted line marks the median normalized read count for all tRNA isodecoders as reference. (C) Log2(fold-change) values between MHV68-infected (MR) versus mock infection from OTTR-seq (green) or DM-tRNA-seq (orange) are consistent for viral-tRNAs (“virtRNA” prefix) and host pre-tRNAs.

Figure 3.. Pre-tRNA accumulation is dependent on MHV68-induced host shut off.

(A) RT-qPCR was performed using total RNA from mock-, MHV68-MR, or MHV68-R443I-infected MC57Gs to detect viral ORF50 (vORF50) and host GAPDH transcripts. Data depicts means +/− SD from three independent experiments relative to the MHV68-MR sample, with p-values calculated using raw ∆Ct values and unpaired t-test. (B) Log2(fold-change) values of MHV68-MR infected vs mock (left) for significantly differentially expressed host mature and premature tRNAs (p<0.05) are graphed alongside the corresponding log2FC for MHV68-R443I vs mock (middle) and MHV68-R443I vs MHV68-MR (right). Horizontal dotted lines indicate a fold change of > or < 2. (C) Normalized read coverage (5’ -> 3’) from mock (left), MHV68-MR (middle), or MHV68-R443I (right) across the Tyr-GTA-1-3 (top) and Gln-CTG-1-1 (bottom) tRNA gene loci. Blue boxes correspond to the gene body of the tRNA, while the horizontal black lines correspond to the 30 bp upstream, 30 bp downstream, and/or intronic regions. (D) Box plot showing the log2(abundance) of three RNA polymerase III transcripts in our sequenced size class (50–200 nt) colored by infection conditions.

Figure 4.. tRNA fragments (tRFs) are induced in a host shutoff-dependent manner.

(A) Log2(fold-change) values of MHV68-MR vs mock (left) for significantly differentially expressed host cytosolic 5’tRFs, 3’tRFs, internal tRFs, and premature-derived tRFs (p<0.05) are graphed alongside the corresponding log2FC for MHV68-R443I vs mock (middle), and MHV68-R443I vs MHV68-MR (right). Horizontal dotted lines indicate a fold change of > or < 2. (B) Volcano plot of log2(FC) values for tRFs from MHV68-MR vs. MHV68-R443I infections, plotted against -log10(pval). tRFs up- or down-regulated more than 2-fold are colored blue for pre-tRFs or black for tRFs generated from mature tRNAs. (C) Normalized read coverage (5’ -> 3’) from mock (left), MHV68-MR (middle), or MHV68-R443I (right) across tRNA gene loci. Blue boxes correspond to the gene body of the tRNA, while the horizontal black lines correspond to the 30 bp upstream, 30 bp downstream, and/or intronic regions.

Figure 5.. Viral TMERs are induced in a host-shutoff-dependent manner.

(A) Schematic of a viral TMER highlighting the 5’ virtRNA and 3’ miRNA regions. (B) Normalized read coverage across the eight viral TMERs encoded within the MHV68 genome from the 50–200nt size selected libraries for mock (top), MHV68-MR (middle), and MHV68-R433I (bottom). (C) Normalized read coverage across TMERs from the 15–50nt size selected libraries for mock (top), MHV68-MR (middle), and MHV68-R433I (bottom). Color bars below indicate either a viral-tRNA feature (green) or a viral-miRNA feature (orange).

Figure 6.. Stem-loop qPCR detection of 5’ tRFs.

(A) Schematic illustrating the specificity of the stem-loop (SL) RT primer for 5’ pre-tRF vs. full-length parental tRNA. (B) Synthetic full-length pre-tRNA-Tyr or 5’ pre-tRF-Tyr were used in known quantities for SL-qPCR using Tyr forward and SL reverse primers. Raw Ct values are shown. (C) Standard- and SL-qPCR was performed using total RNA from mock or MHV68 infected NIH 3T3s to detect the full-length pre-tRNA-Tyr or 5’ pre-tRF-Tyr, respectively. Data depicts means +/− SD from three independent experiments relative to the mock sample, with p-values calculated using raw ∆Ct values and paired t-test. (D) Northern blot using 5’ exon Tyr probes was performed using RNA samples as described in (C). (E) SL-qPCR was performed from four independent experiments to detect 5’ tRF-Arg-TCT and -Gln-CTG as in (C). The sample labeled “ctrl” was reverse transcribed with a SL RT primer for an unrelated tRF species. tDRnamer nomenclature for amplified tRFs is reported in Materials and Methods. ns = p>0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

Figure 7.. The tRNA splicing factors TSEN2 and CLP1 modulate 5’ pre-tRF-Tyr expression during MHV68 infection.

(A) Schematic depicting a pre-tRNA-Tyr transcript (top line) and top three most abundant 5’ pre-tRF-Tyr transcripts detected (purple, blue, and teal lines). The pie charts depict the percentage of detected termini of pre-tRF-Tyr transcripts from mock- and MHV68-MR infected MC57G fibroblasts using OTTR-tRAX. (B) siRNA-mediated knockdown of Tsen2 (siTsen2) and Clp1 (siClp1) or treatment with non-targeting siRNAs (siNT) was followed by mock or MHV68 infection at an MOI=5 for 24 h. siRNA knockdown was confirmed using RT-qPCR using Clp1, Tsen2, and 18S-specific primers. (C) SL-qPCR was used to measure 5’ pre-tRF-Tyr upon siRNA treatment. Data from RT-qPCR and SL-qPCR experiments is depicted as mean +/− SD from four independent experiments relative to the siNT/mock sample, with p-values calculated using raw ∆Ct values and paired t-test. (D) Viral titer of supernatants was measured by TCID50 from three independent experiments, with p-values calculated by one-way ANOVA. ns = p>0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001