Human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation

Chen

et al. 2013

. Furthermore, we found that N L478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N 0 -P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis. Human parainfluenza virus type 3 (HPIV3) is a cytoplasmic, enveloped virus with a nonsegmented negative-strand (NNS) RNA genome that is classified in the Paramyxoviridae family, in the order Mononegavirales. It can cause severe respiratory tract diseases such as bronchiolitis, pneumonia, and croup in infants and young children (1). However, currently no valid antiviral therapy or vaccine is available. Thus, further exploration of its replication mechanism will be helpful in the development of novel therapeutic approaches. The RNA genome of HPIV3 consists of 15,462 nucleotides and is encapsidated by the nucleoprotein (N; 68 kDa) to form a helical nucleocapsid containing N-RNA that has the characteristic herringbone-like structure also observed in other Paramyxoviridae members (2-6). This N-RNA complex serves as a template to interact with the RNA-dependent RNA polymerase (RdRp) complex consisting of a large protein (L; 255 kDa) and a phosphoprotein (P; 90 kDa) cofactor; interaction between N-RNA and RdRp forms an active ribonucleoprotein (RNP) complex that is necessary for transcription and replication (2, 7) to generate six monocistronic mRNAs and an antigenome intermediate. P mRNA encodes a basic protein, designated C, via the translation of a ϩ1 open reading frame of P mRNA, which is responsible for inhibiting viral RNA synthesis as well as counteracting the host interferon signaling pathway (8, 9). A synergic association between the L-P and N-RNA templates would therefore determine the ability of the RNA polymerase complex to transcribe or replicate.Pairs of paramyxoviruses, such as HPIV3 and Sendai virus and canine distemper virus and measles virus, share about 50% nucleotide identity, despite the low level of sequence similarity among known paramyxovirus N genes by sequence comparisons (10)(11)(12). N consists of two major domains that are chemically opposite in nature: a highly conserved N-terminal core (about 80% of the sequence), which forms a globular body, and a hypervariable Cterminal tail (about 20% of the sequence), which extends from the N-terminal body (13). The N terminus contains all of the necessary components for N self-assembly and RNA binding to form N-RNA complex (14-16). Structural assays of the N-RNA complex of some NNS RNA viruses revealed that the RNA is sequestered between the N-and C-terminal lobes of the N-RNA complex (17, 18). The C terminus is mainly responsible for the binding of the N-RNA complex to P (3,(19)(20)(21). Thus, the C terminus is required for the binding of the N-RNA template to the RNA polymerase complex for viral RNA synthesis (22,23). Studies of the nucleocapsid of Sendai virus showed that deletion of the C-terminal fragment abroga...

Section: Discussionmentioning

confidence: 99%

An Amino Acid of Human Parainfluenza Virus Type 3 Nucleoprotein Is Critical for Template Function and Cytoplasmic Inclusion Body Formation

Chen

et al. 2013

“…RSV forms cytoplasmic inclusion bodies (IBs) during infection, as also has been reported for measles virus and human metapneumovirus, also of the family Paramyxoviridae (3)(4)(5). In the case of RSV, the IBs have been shown to contain the RSV nucleoprotein (N), phosphoprotein (P), M2-1 protein, and large polymerase (L) protein (4,6).…”

mentioning

confidence: 91%

p38 and OGT Sequestration into Viral Inclusion Bodies in Cells Infected with Human Respiratory Syncytial Virus Suppresses MK2 Activities and Stress Granule Assembly

Fricke

Koo

Brown

et al. 2013

Respiratory syncytial virus (RSV) forms cytoplasmic inclusion bodies (IBs) that are thought to be sites of nucleocapsid accumulation and viral RNA synthesis. The present study found that IBs also were the sites of major sequestration of two proteins involved in cellular signaling pathways. These are phosphorylated p38 mitogen-activated protein kinase (MAPK) (p38-P), a key regulator of cellular inflammatory and stress responses, and O-linked N-acetylglucosamine (OGN) transferase (OGT), an enzyme that catalyzes the posttranslational addition of OGN to protein targets to regulate cellular processes, including signal transduction, transcription, translation, and the stress response. The virus-induced sequestration of p38-P in IBs resulted in a substantial reduction in the accumulation of a downstream signaling substrate, MAPK-activated protein kinase 2 (MK2). Sequestration of OGT in IBs was associated with suppression of stress granule (SG) formation. Thus, while the RSV IBs are thought to play an essential role in viral replication, the present results show that they also play a role in suppressing the cellular response to viral infection. The sequestration of p38-P and OGT in IBs appeared to be reversible: oxidative stress resulting from arsenite treatment transformed large IBs into a scattering of smaller bodies, suggestive of partial disassembly, and this was associated with MK2 phosphorylation and OGN addition. Unexpectedly, the RSV M2-1 protein was found to localize in SGs that formed during oxidative stress. This protein was previously shown to be a viral transcription elongation factor, and the present findings provide the first evidence of possible involvement in SG activities during RSV infection.

“…The genome is encapsidated by multiple copies of the nucleoprotein (N) to give rise to helical nucleocapsids (7). Additionally, copies of the phosphoprotein (P) interact with N and recruit the L protein, an RNA-dependent RNA polymerase, and the cofactor M2-1 to the viral nucleocapsids (8)(9)(10)(11). Estimates for Sendai virus, a paramyxovirus, show that a virion can contain up to 2,600 copies of N, 300 copies of P, and 50 copies of the L protein (12).…”

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confidence: 99%

“…Nucleocapsids were found within these non-membrane-bound bodies, and therefore, they were proposed to be viral replicative sites. For members of the Pneumoviridae, inclusion body formation upon infection or coexpression of the N and P proteins has been reported (8,29,30).…”

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confidence: 99%

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

Cifuentes-Muñoz

Branttie

Slaughter

et al. 2017

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