Reovirus Replication Protein μ2 Influences Cell Tropism by Promoting Particle Assembly within Viral Inclusions

Abstract

The double-stranded RNA virus mammalian reovirus displays broad cell, tissue, and host tropism. A critical checkpoint in the reovirus replication cycle resides within viral cytoplasmic inclusions, which are biosynthetic centers of genome multiplication and new-particle assembly. Replication of strain type 3 Dearing (T3) is arrested in Madin-Darby canine kidney (MDCK) cells at a step subsequent to inclusion development and prior to formation of genomic double-stranded RNA. This phenotype is primarily regulated by viral replication protein μ2. To understand how reovirus inclusions differ in productively and abortively infected MDCK cells, we used confocal immunofluorescence and thin-section transmission electron microscopy (TEM) to probe inclusion organization and particle morphogenesis. Although no abnormalities in inclusion morphology or viral protein localization were observed in T3-infected MDCK cells using confocal microscopy, TEM revealed markedly diminished production of mature progeny virions. T3 inclusions were less frequent and smaller than those formed by T3-T1M1, a productively replicating reovirus strain, and contained decreased numbers of complete particles. T3 replication was enhanced when cells were cultivated at 31°C, and inclusion ultrastructure at low-temperature infection more closely resembled that of a productive infection. These results indicate that particle assembly in T3-infected MDCK cells is defective, possibly due to a temperature-sensitive structural or functional property of μ2. Thus, reovirus cell tropism can be governed by interactions between viral replication proteins and the unique cell environment that modulate efficiency of particle assembly.

Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

“…We did not observe 12 cells related to cluster 4 (immune response), for the non-infected control, as expected 13 (Fig 2i). These results support the utility of DART-seq to study the single cell 14 heterogeneity in viral genotypes and cellular phenotypes during viral infection. 15 16 DART-seq allows high-throughput paired repertoire sequencing of B lymphocytes 17 18 As a second application of DART-seq, we explored the biological corollary of viral 19 infection, the cellular immune response.…”

“…The high rate of G-to-A transition in the viral transcript could also be 1 secondary to a defect in the fidelity of viral transcription. The T3D strain used in this study 2 has strain-specific allelic variation in the viral polymerase co-factor, µ2, that has been 3 shown to affect the capacity of µ2 to associate with microtubules and the encapsidation 4 of viral mRNAs within capsids 13,14 . To identify distinct host cell populations based on patterns of gene expression, we 1 performed dimensional reduction and unsupervised clustering using approaches 2 implemented in Seurat 15 .…”

“…This is achieved by annealing a double stranded toehold probe with a 3' ssDNA 13 overhang that is complementary to the poly-dT sequence of the Drop-seq primers. The 14 toehold is then ligated to the bead using T4 DNA ligase. Toeholds with a variety of 15 different sequences can be attached to the same primer beads in a single reaction in this 16 manner.…”

Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network

“…In both strains, two species of virus particles were observed, electron dense particles (Figure 2a,b, yellow arrow head) and electron lucent particles (Figure 2a,b, blue arrow head). These particles correspond to genome-containing particles and empty, genome-less particles, respectively (Dales, Gomatos, & Hsu, 1965;Ooms, Jerome, Dermody, & Chappell, 2012). The proportion of genome-containing and genome-less particles was quantified for both strains at 16 hpi using TEM.…”

Electron microscopy: essentials for viral structure, morphogenesis and rapid diagnosis

“…Viral replication is impossible without cells, which make it important to study virus cell interactions [19,60,61] and viral morphogenesis by EM. The common life cycle of viruses comprises adhesion [62], virus host receptor binding [62], entry [63,64] or fusion [65], endocytosis [66], replication [67], assembly [68] and budding [69] (Figures 3 and 4). New viral particles infect other cells and start a new life cycle.…”