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

• Published in: Journal of virology 2018-06, Vol.92 (12), p.e2241-17
• Main Authors: King, Benjamin R, Samacoits, Aubin, Eisenhauer, Philip L, Ziegler, Christopher M, Bruce, Emily A, Zenklusen, Daniel, Zimmer, Christophe, Mueller, Florian, Botten, Jason
• Format: Article
• Language: English
• Subjects: A549 Cells; Cell Line; Genome and Regulation of Viral Gene Expression; Genome, Viral; Genome, Viral - genetics; Humans; In Situ Hybridization, Fluorescence; In Situ Hybridization, Fluorescence - methods; Life Sciences; Lymphocytic choriomeningitis virus; Lymphocytic choriomeningitis virus - genetics; RNA Probes; RNA Probes - genetics; RNA, Viral; RNA, Viral - genetics; Santé publique et épidémiologie; Staining and Labeling; Staining and Labeling - methods; Virus Replication; Virus Replication - genetics
• ISSN: 0022-538X; 1098-5514
• DOI: 10.1128/JVI.02241-17

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 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 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. 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