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. 2018 May 29;92(12):e02241-17.
doi: 10.1128/JVI.02241-17. Print 2018 Jun 15.

Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance During Persistence

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Free PMC article

Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance During Persistence

Benjamin R King et al. J Virol. .
Free PMC article

Abstract

Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule fluorescence in situ hybridization (smFISH) image acquisition and analysis pipeline and examined viral RNA species at discrete time points from virus entry through the late stages of persistent infection in vitro We observed the transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 h of infection, whereas the transcription of glycoprotein mRNA from the S segment antigenome required ∼4 to 6 h. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 h postinfection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by the clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population.IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, lifelong infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication-competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single-cell level. We developed a high-throughput, smFISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses.

Keywords: LCMV; arenavirus; cyclical; gene probes; genome replication and transcription; kinetics; persistence; smFISH.

Figures

FIG 1
FIG 1
LCMV RNA species can be specifically visualized by using multiple, singly labeled oligonucleotide smFISH probes. (A) Overview of the scheme used by arenaviruses to transcribe and replicate their single-stranded, ambisense, bisegmented genome. smFISH probes that recognize the S segment genomic RNA are shown in gray, probes that recognize the S segment genome and GPC mRNA are shown in red, probes that recognize the S segment antigenome and NP mRNA are shown in green, and probes that recognize the L segment antigenome and L mRNA are shown in pink. smFISH probe sets consist of pools of 48 individual 20-mer oligonucleotides, each labeled with a single fluorophore at their 3′ termini. (B) Uninfected cells were stained with a control smFISH probe set specific for MDN1 cellular mRNA labeled with Cy3. (C) Cells either were infected with LCMV at an MOI of 0.01 or, as a control, remained uninfected (mock). Cells were fixed at 24 hpi and stained with a Cy5-labeled smFISH probe set specific for S segment genomic RNA and GPC mRNA. Boxed regions of the cell are magnified and shown in columns labeled “Zoom.” Green arrows indicate example smFISH-stained spots most likely representing single labeled RNAs. Nuclear (hatched lines) and cytoplasmic (solid lines) boundaries are shown in blue. The same intensity levels for a particular probe set were applied to all images of mock- and LCMV-infected cells to permit comparisons. Bars, 10 μm.
FIG 2
FIG 2
smFISH probe sets recognizing viral mRNA species exhibit high signal-to-noise staining. Mock- or LCMV-infected cells (24 hpi) were simultaneously stained with smFISH probe sets specific for either GPC mRNA and the S genome (Cy5) (green) or the S genome only (Alexa Fluor 568) (red). Representative LCMV-infected cells with moderate (A) or high (B) levels of viral RNA as well as a representative mock-infected cell (C) are displayed. Multiple z-stacks spanning the thickness of the cell were acquired, and maximum-intensity projections are displayed. Boxed regions of the cell are magnified and shown in rows labeled “Zoom.” Nuclear (hatched lines) and cytoplasmic (solid lines) boundaries are shown in blue. The same intensity levels for a particular probe set were applied to all images of mock- and LCMV-infected cells to permit comparisons. Bars, 10 μm.
FIG 3
FIG 3
smFISH probe sets recognizing viral mRNA species exhibit high signal-to-noise staining. Shown are signal-to-noise ratios of different smFISH probe sets labeled with the indicated fluorophores. Signal-to-noise ratios were calculated as the average amplitude of detected smFISH spots divided by the standard deviation of the signal in a region of the cell with no detected spots. The signal-to-noise ratio of 20 cells per smFISH probe set labeled with the indicated fluorophore was calculated, and the means and standard deviations are graphed.
FIG 4
FIG 4
Automated detection and quantitation of LCMV RNAs labeled with spectrally distinct fluorophores. (A) Cell nuclei and cytoplasms were automatically segmented by using focus-based projections of DAPI (nuclei) or CellMask green (cytoplasm) z-stacks acquired through the thickness of the cell. Note that pixel intensities of the CellMask green projection displayed here have been log transformed to aid visualization. Nuclear (hatched lines) and cytoplasmic (solid lines) boundaries are shown in white. Bar, 10 μm. (B and C) Maximum-intensity projections of LCMV-infected cells were fixed 24 hpi and stained with smFISH probe sets for the NP mRNA/S antigenome (Cy5) (green) and GPC mRNA/S genome (A568) (red). The boxed region of each cell is magnified and shown in the row labeled “Zoom.” Cells were segmented based on DAPI and CellMask green staining (see panel A), and spots were detected and localized in 3D by using FISH-quant. Individually detected RNAs are circled in green (NP mRNA/S antigenome) or red (GPC mRNA/S genome). The “Spots Only” column shows only the positions of the detected spots in relation to the cell boundaries defined by segmentation. Nuclear (hatched lines) and cytoplasmic (solid lines) boundaries are shown in blue. The same intensity levels for a particular probe set were applied to both images of LCMV-infected cells to permit comparisons. Bar, 10 μm. (D) Scatter plot showing the relationship between the fluorescence intensity in the smFISH channel in the maximum-intensity projection of smFISH images and the number of smFISH spots detected by FISH-quant for LCMV-infected cells fixed at 24 hpi and stained with the Cy5-labeled smFISH probes specific for the NP mRNA/S antigenome.
FIG 5
FIG 5
Transcription of NP and L genes is detectable soon after infection, while GPC transcription occurs exclusively after a several-hour lag. Cells were infected with LCMV at an MOI of 0.1, fixed at various times following infection, and stained for NP mRNA (green) using a Cy5-labeled NP mRNA/S antigenome probe set and GPC mRNA (red) using an A568-labled GPC mRNA/S genome probe set (A) or for NP mRNA (green) using an A568-labeled NP mRNA/S antigenome probe set and L mRNA (magenta) using a Quasar 670-labeled L mRNA/L antigenome probe set (B). Note that for the time points shown (prior to 8 hpi), genomic and antigenomic RNAs are not detectable by smFISH probe sets with exclusive specificity for these RNAs (data not shown). Therefore, spots detected in this figure are presumed to represent only the mRNAs, but not the genome or antigenome, targeted by each respective probe set. Nuclear (hatched lines) and cytoplasmic (solid lines) boundaries, as determined by CellProfiler, are shown in blue. Identified spots are outlined by circles, which are green for NP mRNA, red for GPC mRNA, and magenta for L mRNA. The same intensity levels for a particular probe set were applied to all images of mock- and LCMV-infected cells across the time course to permit comparisons. Representative maximum-intensity projections from 1 of 2 independent experiments are shown. Bars, 10 μm.
FIG 6
FIG 6
Transcription of NP and L genes is detectable immediately upon infection, while GPC transcription occurs exclusively after a several-hour lag (Fig. 5). (A and B) Box plots representing the number of viral RNAs detected in cells at early time points following infection with LCMV (Fig. 5). (C and D) Stacked bar graphs showing the proportions of cells expressing RNAs detected by one, both, or neither viral RNA smFISH probe set. Between 620 and 1,316 cells were examined at each time point. In each case, RNAs identified by specific probe sets are designated by color. Note that for time points prior to 8 hpi, genomic and antigenomic RNAs are not detectable by smFISH probe sets with exclusive specificity for these RNAs (data not shown). Therefore, spots detected before 8 hpi are presumed to represent only the mRNAs, but not the genome or antigenome, recognized by each respective probe set. Spots detected at 8 hpi or later are presumed to be a mixture of all RNAs recognized by a particular probe set (e.g., mRNA and the genome or antigenome).
FIG 7
FIG 7
Peak viral RNA replication and transcription occur at 36 hpi and are slowly lost from infected cells over the following days. Cells were infected with LCMV at an MOI of 0.01, fixed at various times following infection, and stained by using smFISH probe sets specific for the NP mRNA/S antigenome (Cy5) (green) and the GPC mRNA/S genome (A568) (red) (A) or for the NP mRNA/S antigenome (A568) (green) and the L mRNA/L antigenome (Quasar 670) (magenta) (B). Representative maximum-intensity projections of fields of infected cells at various time points from 1 of 2 independent experiments are shown. Each probe set is shown in its own row to highlight the difference in the levels to which these RNAs accumulate. The same intensity levels for a particular probe set were applied to all images of mock- and LCMV-infected cells across the time course to permit comparisons. Bars, 10 μm.
FIG 8
FIG 8
Peak viral RNA replication and transcription occur at 36 hpi and are slowly lost from infected cells over the following days (Fig. 7). (A and B) Box plots representing the numbers of mRNAs detected in cells at time points during the peak period of LCMV infection. (C and D) Stacked bar graphs showing the proportions of cells expressing RNAs detected by one, both, or neither viral smFISH probe set. Between 480 and 1,659 cells were examined at each time point. RNAs identified by specific probe sets are designated by color.
FIG 9
FIG 9
Cyclic periods of infectious virus particle release and antigen expression during persistence. Cells were infected with LCMV at an MOI of 0.01. (A) Supernatants from infected cells were collected at the indicated time points postinfection, the titers were determined, and the data are presented as mean PFU per milliliter ± standard deviations from 3 independent experiments. (B and C) Cells were fixed at the indicated time points following infection and stained by using smFISH probe sets specific for the NP mRNA/S antigenome (A568) (red), the GPC mRNA/S genome (Cy5) (magenta), and NP (Alexa 488) (green) (B) or GPC (Alexa 488) (green) (C). A single z-slice of a representative field of infected cells at various time points is shown. Each antibody or probe set is shown in its own row, and the same intensity levels were applied to all images of mock- and LCMV-infected cells across the time course to permit comparisons. Bars, 10 μm.
FIG 10
FIG 10
Cyclic periods of viral RNA production and viral RNA loss occur during persistence. (A and D) Cells were infected with LCMV at an MOI of 0.01, fixed at the indicated time points following infection, and stained by using smFISH probe sets specific for the NP mRNA/S antigenome (Cy5) (green) and the GPC mRNA/S genome (A568; red) (A) or for the NP mRNA/S antigenome (A568; green) and the L mRNA/L antigenome (Quasar 670) (magenta) (D). Representative maximum-intensity projections of fields of infected cells at various time points from 1 of 2 independent experiments are shown. Each probe set is shown in its own row to highlight the difference in levels to which these RNAs accumulate. The same intensity levels for a particular probe set were applied to all images of mock- and LCMV-infected cells across the time course to permit comparisons. Bars, 10 μm. (B and E) Line graphs showing the average numbers of the indicated viral RNAs detected in cells at time points during the persistent phase of LCMV infection. (C and F) Stacked bar graphs showing the proportions of cells expressing RNAs detected by one, both, or neither viral smFISH probe set. Between 316 and 1,218 cells were examined at each time point.

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