Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

Abstract

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positivesense RNA (ϩRNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.KEYWORDS HMPV, pneumovirus, inclusion bodies, replication H uman metapneumovirus (HMPV) is a pathogen that affects all age groups worldwide, with an increased risk of severe disease being found in immunocompromised individuals, children under the age of 5 years, and the elderly (1-4). Together

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Direct cell-to-cell transmission of respiratory viruses: The fast lanes

“…Instead, we hypothesize that direct cell-to-cell spread of infection might occur in the form of partially assembled viral particles ( Fig 2b ) or nucleocapsids ( Fig 2c ). Interestingly, structures similar to inclusion bodies, which recent studies strongly suggest are organelles involved in replication for several respiratory viruses [ 23 , 24 ], have been observed moving through intercellular extensions [ 5 ]. The potential transfer of a replicative inclusion body from one cell to another ( Fig 2d ) would allow the virus to bypass the first critical hours of infection, and examination of this intriguing hypothesis could alter our understanding of viral spread.…”

Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

“…Similar structures called Negri bodies were initially observed upon rabies virus (RABV) infection and constituted a signature for the diagnosis of the infection ( 6 , 7 ). Since then, it appeared that IBs are a hallmark of infection for many other MNVs such as vesicular stomatitis virus (VSV) ( 8 ), measles virus (MeV) ( 9 ), metapneumovirus (MPV) ( 10 ), Ebola virus ( 11 ), Marburg virus ( 12 ), Nipah virus ( 13 ), and parainfluenza virus (PIV) ( 14 , 15 ). It was shown that these structures are viral factories where all the viral proteins of the polymerase complex concentrate to perform the replication and transcription of the viral genome ( 16 ).…”