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 the biological function for tRNA cleavage products, including the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus 68, 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. 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, which regulates tRNA splicing among other RNA processing events, is required for efficient MHV68 replication. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing.IMPORTANCEDiverse conditions of infection and cellular stress incite the cleavage of transfer RNAs (tRNAs), leading to the formation of tRNA fragments (tRFs) that can directly regulate gene expression. In our study of gammaherpesviruses, such as murine herpesvirus 68 and human oncogenic Kaposi sarcoma-associated herpesvirus, we discovered that tRNA regulation and cleavage are key components of gene reprogramming during infection. We present the first in-depth profile of tRF generation in response to DNA virus infection, using state-of-the-art sequencing techniques that overcome several challenges with tRNA sequencing. We present several lines of evidence that tRFs are made from newly transcribed premature tRNAs and propose that this may be a defining characteristic of tRNA cleavage during infection. Finally, we show that tRNA splicing machinery is involved with the formation of some MHV68-induced tRFs, with a key regulator of splicing, CLP1, required for maximal viral titer. Taken together, we posit that tRNA processing may be integral to the elegant shift in gene expression that occurs during viral takeover of the host cell.

Keywords: herpesviruses; tRNA; tRNA fragments; tRNA splicing; viral tRNA; virology.

Fig 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.

Fig 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 mouse fibroblasts at an MOI = 5 for 24 h. (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.

Fig 3

Pre-tRNA accumulation is dependent on MHV68-induced host shutoff. (A) RT-qPCR was performed using total RNA from mock-, MHV68-MR, or MHV68-R443I-infected MC57Gs (MOI = 5 for 24 h) to detect viral ORF50 (vORF50) and host GAPDH transcripts. Data depict means ± SD from three independent experiments relative to the MHV68-MR sample, with P values calculated using raw ΔCt values and unpaired t-test. (B) Supernatants from MHV68-MR and MHV68-R443I-infected MC57Gs (MOI = 5 for 24 h) were titered by TCID₅₀. (C) The complete set of significantly differentially expressed host mature and premature tRNAs upon MHV68-MR infection (P < 0.05; MHV68-MR vs Mock) are plotted alongside their log2(fold change) values. Log2(fold change) values for MHV68-MR-infected versus mock (left) are graphed alongside the corresponding log2FC for MHV68-R443I versus mock (middle) and MHV68-R443I versus MHV68-MR (right). Vertical dotted lines indicate a fold change of >2 or <2. (D) 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. (E) Box plot showing the log2(abundance) of three RNA polymerase III transcripts in our sequenced size class (50–200 nt) colored by infection conditions.

Fig 4

tRNA fragments (tRFs) are induced in a host shutoff-dependent manner. (A) The complete set of significantly differentially expressed host tRFs (host cytosolic 5′tRFs, 3′tRFs, internal tRFs, and premature-derived tRFs) upon MHV68-MR infection (P < 0.05; MHV68-MR vs Mock) are plotted alongside log2(fold change) values. Log2(fold change) values of MHV68-MR versus mock (left) are graphed alongside the corresponding log2FC for MHV68-R443I versus mock (middle), and MHV68-R443I versus MHV68-MR (right). Vertical dotted lines indicate a fold change of >2 or <2. (B) Volcano plot of log2(FC) values for tRFs from MHV68-MR versus MHV68-R443I infections, plotted against −log10(P value). tRFs up- or downregulated more than twofold 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. (D) Significantly differentially expressed host tRFs that appear to be derived from mature tRNAs (5′tRFs, 3′tRFs, and internal tRFs) upon MHV68-MR infection (P < 0.05; MHV68-MR vs Mock) are plotted. The log2(fold change) for both the parental mature tRNA (black bars) and the tRFs (orange bars) are graphed for comparison. nd = not detected in sequence run. There is no correlation between changes in mature tRNA and their cognate tRFs.

Fig 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 50–200 nt size selected libraries for mock (top), MHV68-MR (middle), and MHV68-R433I (bottom). (C) Normalized read coverage across TMERs from the 15–50 nt 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).

Fig 6

Stem-loop (SL) qPCR detection of 5′ tRFs. (A) Schematic illustrating the specificity of the SL RT primer for 5′ pre-tRF versus 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 and D) Standard- and SL-qPCR was performed using total RNA from mock- or MHV68-infected (MOI = 5 for 24 h) NIH 3T3s (C) or MC57Gs (D) to detect the full-length pre-tRNA-Tyr or 5′ pre-tRF-Tyr, respectively. 18S or U6 primers were used as endogenous controls. Data depict means ± SD from three to five independent experiments relative to the mock sample. (E) Northern blot using 5′ exon Tyr probes was performed using mock- or MHV68-infected (MOI = 5 for 24 h) MC57G RNA samples. (F) SL-qPCR was performed from four independent experiments to detect 5′ pre-tRF-Tyr produced in NIH 3T3 during infection (MOI = 5 for 24 h). Data depict means ± SD relative to the 0 h time point of each experiment. (G) SL-qPCR was performed from four independent experiments to detect 5′ tRF-Arg-TCT and -Gln-CTG using mock- or MHV68-infected (MOI = 5 for 24 h) NIH 3T3 RNA samples. The sample labeled “ctrl” was generated with RNA reverse transcribed with a SL RT primer for an unrelated tRF species. tDRnamer (21) nomenclature for amplified tRFs is reported in Materials and Methods. All P values were calculated using raw ΔCt values and t-test. ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig 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 are depicted as mean ± SD from four independent experiments relative to the siNT/mock sample, with P values calculated using raw ΔCt 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.

Fig 8

CLP1 is required for efficient MHV68 replication. (A) siRNA-mediated knockdown of Tsen2 (siTsen2) and Clp1 (siClp1) or treatment with non-targeting siRNAs (siNT) was followed by mock or MHV68-MR infection at an MOI = 5 for 24 h. Cells were analyzed for GFP expression by flow cytometry. (B) Supernatants from the conditions in (A) were titered by TCID₅₀. (C) Stable cell lines expressing pEF1A:empty (empty), pEF1A:Clp1, or pEF1A:Tsen2 were validated by RT-qPCR using Clp1, Tsen2, and 18S-specific primers. (D) Stable cell lines expressing pmCherry (empty), pEF1A:Clp1, or pEF1A:Tsen2 were infected at both low (MOI = 0.05) and high (MOI = 5) MOIs and titered at 3 dpi or 24 hpi, respectively, by TCID₅₀. (E and F) siRNA-mediated knockdown of Clp1 (siClp1) or treatment with non-targeting siRNAs (siNT) was followed by MHV68-MR infection at an MOI = 5. Samples were collected at indicated time points and analyzed by TCID₅₀ (E) or RT-qPCR using viral ORF50, viral gB, and 18S-specific primers (F). All P values were calculated using raw ΔCt or TCID₅₀/mL values and one-way ANOVAs or unpaired t-test. ns = P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.