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Open access peer-reviewed chapter

Respiratory Syncytial Virus

Written By

Sattya Narayan Talukdar and Masfique Mehedi

Submitted: 24 February 2022 Reviewed: 01 April 2022 Published: 28 July 2022

DOI: 10.5772/intechopen.104771

RNA Viruses Infection IntechOpen
RNA Viruses Infection Edited by Yogendra Shah

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RNA Viruses Infection

Edited by Yogendra Shah

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Abstract

Respiratory Syncytial Virus (RSV)-driven bronchiolitis is one of the most common causes of pediatric hospitalization. Every year, we face 33.1 million episodes of RSV-driven lower respiratory tract infection without any available vaccine or cost-effective therapeutics since the discovery of RSV eighty years before. RSV is an enveloped RNA virus belonging to the pneumoviridae family of viruses. This chapter aims to elucidate the structure and functions of the RSV genome and proteins and the mechanism of RSV infection in host cells from entry to budding, which will provide current insight into the RSV-host relationship. In addition, this book chapter summarizes the recent research outcomes regarding the structure of RSV and the functions of all viral proteins along with the RSV life cycle and cell-to-cell spread.

Keywords

  • RSV
  • RNA virus
  • RNA genome
  • replicative cycle
  • fusion protein
  • cell-to-cell spread
  • filopodia

1. Introduction

Human respiratory syncytial virus (RSV), despite being a human virus, was first isolated in 1955 from a chimpanzee with respiratory illness [1]. Since its first discovery, it did not take long to isolate RSV from infants with respiratory diseases. Indeed, serological studies verified the existence of RSV infection in infants and children [2, 3]. Now, RSV infection is a prominent cause of lower respiratory tract diseases (bronchiolitis and pneumonia) and hospitalization in children worldwide [4]. According to the most recent virus taxonomy, RSV now belongs to a new family Pneumoviridae of the order Mononegavirales [5].

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2. RSV virion

RSV is an enveloped and cytoplasmic virus with non-segmented, negative-sense, single-stranded RNA genome [6]. RSV virions are known to bud out on the infected cell surface. The filamentous virion is up to 12 μm in length and 60 to 200 nm in diameter (Figure 1) [7, 8, 9]. RSV virions can be irregular-shaped spherical particles with a diameter ranging from 100 to 350 nm. Both filamentous and spherical virus particles mostly remain cell-associated [9].

Figure 1.

RSV virion. A photomicrograph of an RSV filamentous virion. The image was taken under electron microscope.

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3. RSV strains

There are two RSV strains as RSV A and RSV B and are categorized on basis of genetic and antigenic differences [10]. However, mostly extensive antigenic and nucleotide sequence variation was observed between RSV A and RSV B, however, genetic as well as antigenic variability was also studied within the individual groups of RSV [11]. Multiple studies demonstrated the differences in viral replication between these two groups; specifically, RSV A replicated to higher titers than RSV B viruses in both cell culture and animal models [12, 13, 14, 15, 16]. In addition, RSV A infection is more virulent and severe than RSV B [17].

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4. RSV RNA and proteins

The RSV genome is a single-stranded, negative-sense RNA whose length is ranging from 15,191 to 15,226 nucleotides [9]. The RSV genome contains ten genes in the order 3′-NS1-NS2-N-P-M-SH-G-F-M2-L-5′ that are transcribed sequentially into 10 independent messenger RNAs (mRNAs) (Figure 2). Each RSV mRNA encodes a single major protein except for M2, which encodes two separate open reading frames (ORF) for M2–1 and M2–2 proteins, respectively [9, 18, 19, 20]. The M2–1 and M2–2 ORF are located in the upstream and downstream parts of the mRNA, respectively [9]. Like many RNA viruses, RSV brings ribonucleoprotein (RNP) complex as a piece of transcriptional machinery for its genome transcription and replication inside the infected cell cytoplasm. The RNP complex consists of the viral genome, nucleoprotein (N), phosphoprotein (P), and RNA-dependent RNA polymerase [L) [21].

Figure 2.

Schematic of an RSV genome. RSV genome is a negative-sense non-segmented single-stranded RNA. The genome contained 10 genes oriented from 3′ end: NS1, NS2, N, P, M, SH, G, F, M2 (M2–1 and M2–2), and L.

4.1 Nucleoprotein (N)

The RNA genome is wrapped by N (391 amino acids) to create a nuclease-resistant, helical RNP complex called nucleocapsid (NC), and it functions as the template for both replication and transcription [22, 23]. RSV virus genome for replication does not follow the “rule of six” [24], which is common to most paramyxoviruses [25]. The three-dimensional (3D) crystal structure revealed a decameric, ribonucleoprotein complex of N protein and RNA with 3.3 A° resolution and suggested N protein can function as a helicase to separate temporary double-stranded RNA during RNA synthesis [23]. As a decameric structure, every N subunit has a core region comprising two domains, N-terminal and C-terminal, which are linked by a hinge region and the RNA genome turns inside a basic surface groove located at the interface of N-terminal/C-terminal; specifically, every N subunit interacts with seven ribonucleotides of RNA [23].

4.2 RNA-dependent RNA polymerase (L)

The L protein (2165 amino acids) has three enzymatic domains including RNA-dependent RNA polymerase (RdRp) domain, polyribonucleotidyl transferase domain which is essential for capping located in its N-terminal, and methyltransferase domain which is necessary for cap methylation located in C-terminal [26, 27, 28, 29]. Viral mRNA undergoes a post-transcriptional modification before translation and methyltransferase plays a significant role by catalyzing the methylation of cap structure at both N7- and 2′-O-positions because N7-methylation is vital for viral RNA translation and 2′-O-methylation is important for hiding viral RNA from the innate immunity system [30].

4.3 Phosphoprotein (P)

The P protein (241 amino acids) is a homotetrameric protein consisting of N-terminal domain, oligomerization domain, and C-terminal domain and it functions as a cofactor of RdRp and plays a significant role in transcription and replication by networking with other RSV proteins [31, 32, 33, 34, 35]. P protein functions as a multimodular adaptor for RNA synthesis by interacting with N-RNA, L, and M2–1 [36]. P can act as a chaperone for newly synthesized N (N0) protein by forming an N0-P complex that prevents the association of N0 with host RNA [37]. This protein is heavily phosphorylated by host kinase enzymes and it has 41 serine and threonine residues as potential phosphorylation sites; specifically, phosphorylation at residues T105, T188, T210, and S203 are essential for replication, and phosphorylation at residue S156 is vital for viral RNA synthesis [38].

4.4 RSV glycoproteins

As an enveloped virus, the RSV lipid envelope contains three transmembrane glycoproteins including a fusion (F) protein, an attachment glycoprotein (G), and a small hydrophobic (SH) protein; F and G proteins are essential for viral attachment and entry whereas SH protein is less likely involved in viral entry and budding [39, 40].

4.4.1 Fusion (F) protein

Fusion protein is a type 1 transmembrane protein (574 amino acids including a cytoplasmic tail domain of approximately 24 residues) involved in viral entry and assembly [39, 41]. Initially, F protein is synthesized as F0 protein and subsequently, F0 undergoes post-translational modification with multiple N-linked glycosylations depending on RSV strains [42]. To obtain fusion competence, precursor F0 protein (approximately 68–75 KDa) undergoes proteolytic cleavage by furin-like protease which cleaves two polybasic sites and removes a glycosylated peptide of 27 amino acids (Peptide 27 or Pep27) [43, 44]. This cleavage process occurs in the trans-Golgi network and then fusion protein transport to plasma membrane generating two subunits: one is amino-terminal F2 subunit (approximately 15–20 KDa) and another is carboxy-terminal F1 subunit (approximately 50–55 KDa) [45, 46]. A heterodimeric protomer is formed by F1 and F2 subunits covalently connected by disulfide bonds and three protomers combinedly form the matured trimeric form of F protein [47]. After trimerization, F protein exists as a prefusion conformation remaining approximately 12 nm above the membrane of the virus [48]. This prefusion conformation is not a stable form and undergoes a refolding process [6, 49]. This refolding process creates a more stable post-fusion conformation of F protein remaining approximately 17 nm above the viral membrane [50, 51]. The sequence difference of F ectodomains is almost 5% between RSV A and RSV B and therefore, F protein undergoes less antigenic drift and gets preference for suitable vaccine candidates [52].

4.4.2 Attachment glycoprotein (G)

In RSV-infected cells, G protein can exist in two forms; one is a membrane-bound form responsible for viral attachment and another is a secreted isoform responsible for immune evasion [53, 54]. The membrane-bound form (298 amino acids) is a type 2 integral membrane protein [55]. G protein has an amino-terminal cytoplasmic domain and a hydrophobic transmembrane domain; moreover, its ectodomain which undergoes post-translational modification with 4–5 N-linked glycans and 30–40 O-linked glycans, has two mucin-like regions and heparin-binding domains [55, 56, 57]. The translation of secreted G protein starts at an alternative AUG (Met48) located in the transmembrane domain allowing the ectodomain to secrete from the cell [58]. Both membrane-bound and secreted forms of G proteins are thought to be involved in RSV pathogenesis [59]. The higher variation of the mucin-like domain caused two subtypes of RSV: RSV A and RSV B [60].

4.4.3 Small hydrophobic (SH) protein

SH glycoprotein is a small transmembrane protein (64 amino acids for RSV A and 65 amino acids for RSV B) attached by a hydrophobic signal-anchor sequence closer to the N-terminal with extracellular C-terminal orientation; in addition, this protein is considered as less immunogenic because of smaller size and lower abundance during RSV infection [61]. It can exist in several forms including full-length form or post-translational modified form by glycosylation and phosphorylation [62]. Although its function is not clearly understood like other glycoproteins, several studies suggested SH protein can play an auxiliary role during viral fusion along with F glycoprotein; however, SH protein is not crucial for viral entry and syncytium formation [63, 64, 65]. SH protein primarily amasses in the lipid raft membrane of the Golgi complex and endoplasmic reticulum; however, lower levels of SH protein are associated with the envelope of filamentous virus [40]. SH protein did not play an essential role during viral replication in cell culture but SH-deleted RSV infection caused 10-fold lower titers in animal models [39, 66]. It can induce membrane permeability and form pentameric ion channels suggesting its role as viroporins which are short (approximately 100 amino acids) membrane proteins forming oligomers to act as ion channels [67]. Moreover, SH protein is essential to activate the NLRP3 inflammasome [68, 69]. The role of SH protein on apoptosis is not clear because RSV infected A549 cells produced TNF-α and cells were not sensitive to TNF-α-induced death but cells demonstrated a higher level of apoptosis after SH-deleted RSV infection indicating that RSV SH protein may affect the TNF-α pathway resulting in apoptosis delay by an alternative mechanism [70].

4.5 RSV matrix proteins (M and M2)

RSV has two matrix proteins including M protein and M2 protein [58].

4.5.1 M protein

M protein (256 amino acids) is a non-glycosylated protein located in the innermost part of the viral envelope [71]. It is the main protein responsible for viral assembly and budding by interacting with the cell membrane, viral envelope, and viral nucleocapsid [72, 73]. M protein has a zinc finger domain, two clusters of basic amino acids indicating a nuclear localization signal and two nuclear export signals and its N-terminal has lower hydrophobicity; in contrast, C-terminal has higher hydrophobicity [74]. M protein contains multiple phosphorylation sites and undergoes phosphorylation during infection but it is unclear whether these phosphorylations control its function [75]. During the early phase of infection, M protein is present in the host nucleus and inhibits host cellular transcription [76]. During the late phase of infection, M protein is mostly cytoplasmic, interacts with nucleocapsid, and inhibits the activity of viral transcriptase [77]. M protein is located in the cytoplasmic part of the plasma membrane-associated with the lipid rafts along with G and N proteins implying that lipid rafts can function as a platform for the assembly and budding of RSV [73]. M protein is active in a dimer form and the conversion of M-M dimer to oligomer is essential for viral assembly because the interference of dimer formation reduces viral filament maturation and budding [21].

4.5.2 M2 (M2-1 and M2-2) protein

M2–1 and M2–2 are nucleocapsid associated proteins [78]. RSV M2 gene has two overlapping ORFs as M2–1 and M2–2 [79]. The recent crystal structure of the M2–1 (194 amino acids) protein has revealed its native tetrameric form with 2.5 Å resolution and each of its monomers contains three domains including zinc-binding, oligomerization, and core domains [80, 81]. M2–1 functions as a transcriptional anti-terminator and processivity factor [79, 82]. M2–1 did not affect genome and antigenome synthesis indicating that M2–1 is not involved in RNA replication [79, 83]. M2–2 protein (90 amino acids) acts as a regulatory factor switching from transcription to RNA replication because mRNA accumulation was intensely higher after 12–15 hours of infection and then flattened in case of wild-type virus infection but M2–2 knockout virus infection showed continued accumulation [80]. Another study showed M2–2 protein could negatively regulate transcription and positively modulate RNA replication because recombinant RSV infection without NS1 and M2–2 protein demonstrated ten times lower viral growth kinetics in the upper respiratory tract of infants [84].

4.6 RSV nonstructural (NS) proteins (NS1 and NS2)

RSV NS proteins including NS1 (139 amino acids) and NS2 (124 amino acids) play a crucial role in interfering with host innate immunity by forming a “Nonstructural degradosome complex” which can act as a proteasome-like complex that disintegrates a massive number of proteins involved in the innate immune system [85, 86]. Infection with NS1 and NS2 single- and double-gene-deleted RSV demonstrated that both proteins function individually and jointly to accomplish the complete inhibitory effect on type I and III IFNs whereas NS1 has a more individual function [87, 88]. Both NS1 and NS2 target retinoic acid-inducible gene I (RIG-I) like receptors (RLRs), which are considered as host pattern recognition receptors for RIG-I and melanoma differentiation-associated gene 5 (MDA5) [89]. Both NS1 and NS2 induce multiple chemokines and cytokines like RANTES, IL-8, TNFα during viral infection [90]. RIG-I activation by ubiquitination is vital for stimulating antiviral response and tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is essential for RIG-I activation [91]. NS1 protein inhibits RIG-I ubiquitination by interacting with TRIM25 and eventually suppresses type-I interferon (IFN) signaling [92]. Cytosolic NS1 can go to the host nucleus and interacts with the gene regulatory domains of immune response genes, which can control gene transcription and eventually modulates host response against RSV infection [93]. NS1 localized to mitochondria inhibits type-I interferon (IFN) signaling by binding with mitochondrial antiviral signaling protein (MAVS) because the MAVS-RIG-1 complex is essential for type-I IFN activation [94]. NS1 also stimulates miR-29a expression, which affects mRNA coding for interferon alpha/beta receptor 1 (IFNAR1) [95]. NS1 enhances autophagy by the mTOR pathway, which is beneficial for RSV replication but inhibits apoptosis and multiple inflammatory cytokines and IFN-α [96]. Recombinant RSV (NS-deficient) infection showed that mostly NS1 (partially NS2) inhibits the maturation of Dendritic cells, which in turn activates B and T cell responses [97]. NS1 can also inhibit the anti-inflammatory effect of glucocorticoids [98]. The recent X-ray crystal structure of NS2 reveals that it has a unique fold that allows to target molecules different from NS1 and activates distinct IFN antagonism pathway compared to NS1 [99]. Recombinant RSV virus without NS2 showed lower viral growth indicating the role of NS2 in viral replication by evading host immunity [100]. The increased level of IFNβ was not as high when recombinant RSV without NS1 or NS1/NS2 were applied suggesting that both NS1 and NS2 work together for interferon signaling suppression [84]. NS2 also plays a significant role in NF-κB activation, which can initiate a cascade by binding transcription promoters of several proinflammatory cytokines along with IRF-3 and IFN-α/β [90]. In addition to innate immunity, NS2 interferes with adaptive immunity by suppressing CD8+ T-cell responses as a consequence of controlling type 1 IFN [101]. Mostly NS2 along with NS1 play a role in delaying apoptosis, which can enable prolonged RSV replication by activating 3-phophoinositide-dependent protein kinase (PDK)-RAC serine/threonine-protein kinase-glycogen synthase kinase (GSK) pathway [102]. In addition, NS2 plays a significant role in modulating cell morphology, which causes the shedding of infected cells and the spreading of RSV virions [103].

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5. Replicative cycle of RSV

5.1 Entry

RSV infection mostly occurs in the apical side of ciliated cells and type 1 pneumocyte; however, several reports suggested the presence of RSV RNA in the extrapulmonary sites and fluids, but more investigations are required [104, 105, 106, 107]. RSV entry has two major phases; the first step is virion attachment to the host cell and the next step is the fusion of viral and host cell membranes in which host factors can involve in both or any individual phases [52]. Heparin-binding domain located between mucin-rich domains of G protein interacts with the unbranched disaccharide polymers specifically glycosaminoglycans (GAGs) connected to transmembrane proteins on the cell surface for the attachment observed in multiple cell culture studies [108, 109, 110]. Variation of G protein lacking heparin-binding domain showed viral attachment indicating the involvement of other regions of G protein during attachment [108]. Negatively charged regions of heparin sulfate contribute mostly and iduronic acid-containing GAG contributes minimally to the attachment [111, 112, 113]. Heparan sulfate proteoglycans (HSGP) act as the receptor for G protein in cell lines; however, recombinant RSV without G protein showed its infectivity; in contrast, HSGP does not express in ciliated epithelial cells, but G protein is still essential for infection in vivo [114, 115, 116]. However, the apical side of ciliated cells, which is the major site of RSV infection lack heparin sulfate indicating the involvement of other host factors, specifically, fractalkine receptor CX3C-chemokine receptor 1 (CX3CR1) bind to CX3C motif of G protein for the attachment [117, 118]. CX3CR1 expressed on the ciliated cells, acts as the receptor of G protein by interacting with its CX3C motif and mutations in the CX3C motif of G protein reduces RSV infection in vivo [117, 119, 120, 121]. F protein is involved in the viral attachment because RSV lacking G and SH proteins grows in cell culture studies and it interacts with heparin sulfate like G protein causing attachment and subsequent infection [63, 122, 123]. Almost 50% infection was observed after heparinase treatment and without GAG synthesis while RSV has F protein suggesting the interaction of F with other host factors; particularly, F protein facilitates entry by interacting with intercellular adhesion molecule 1, insulin-like growth factor 1, epidermal growth factor receptor, and nucleolin [124, 125, 126, 127]. Host and viral membrane then fuse after attachment so that viral particles can enter the cytoplasm and this fusion process is pH-independent and insensitive to lysosomal acidification [128, 129]. RSV infection induces an actin mediated rearrangement followed by plasma membrane blebbing and excess fluid uptake causing internalization of viral particles in a Rab5 positive macropinisome and this endocytic entry depends on the activation of F protein by a second proteolytic cleavage catalyzed by furin-like enzymes after endocytosis observed in A549 cell [130].

5.2 Transcription and replication

RSV replication and transcription are dependent on viral components including viral RNA, N, P, L, and M2–1 [131]. RSV utilizes its own machinery (RNP complex) to replicate in the host cytoplasm [132]. Inclusion body formation is a hallmark of RSV infection produced by multiple viral proteins including N, P, L, and M2–1 and this cytoplasmic structure is increased with RSV infection in epithelial cells [72, 133, 134]. Specifically, N and P proteins are important for inclusion body formation because the expression of these proteins with or without RSV infection showed inclusion body formation [135]. P protein can hijack host cell machinery by forming a complex with host phosphatase (PP1) and this P-PP1 complex dephosphorylates M2–1, as a result, P protein can recruit M2–1 protein in the inclusion body to facilitate viral RNA synthesis [136]. M protein is also reported to localize in inclusion bodies mediated by M2–1 protein [137]. The inclusion body is thought to be the first place where M protein interacts with the ribonucleoprotein complex and M protein is involved in the release of RNP from inclusion bodies towards budding [138]. Host actin cytoskeleton and Hsp70 proteins are also observed in inclusion bodies, but their role is not clear yet and they perhaps facilitate viral machinery [139]. RSV infection causes vigorous stress on the host cell resulting formation of cytoplasmic stress granules, which are different from cytoplasmic inclusion bodies and these stress granule formations facilitates viral replication [140].

Both viral RNA replication and mRNA transcription start from the same single promoter in leader (le) region (44-nucleotide long) at the 3′ end of RSV genome and it produces methyl-guanosine capped and polyadenylated mRNA during transcription and antigenome during replication [20, 141, 142, 143]. Each RSV gene has two conserved cis-acting elements including a gene start (gs) signal at the beginning and a gene end (ge) signal at the end [144]. The promoter of leader (Le) region at the 3′ end of RSV genome has two initiation sites, one is at position +1 or 1 U required for replication and another one is at position +3 or 3C required for transcription [145]. 9 out of 10 gs signaling sequences are highly conserved whereas the tenth one has minimal sequence difference in RSV genome [19]. During transcription, both gs and ge signaling sequences play significant role, specifically, gs signal provides direction to RNA-dependent RNA-polymerase (RdRp) for initiating RNA synthesis and ge signal provides direction to RdRp to polyadenylate and release the mRNA [146, 147]. Then RdRp connected to the template can initiate transcription again at the next gs signal and this process persists along RSV genome [144]. During replication, RdRp attaches a similar promoter sequence in le region, but it ignores ge signal and continues to proceed throughout the genome to produce an antigenome, which is a full-length positive-sense complement of RSV genome [145]. Viral genome and antigenome RNA are encapsidated in RSV nucleoprotein whereas viral mRNAs are not encapsidated [145]. Every nucleoprotein monomer interacts with 7 nucleotides of viral RNA and this complex forms a helical nucleocapsid acting as a template for the next RNA synthesis. This encapsidation is thought to increase RdRp activities to override ge signal during replication, therefore, encapsidation is the distinguishing factor between replication and transcription [23, 148, 149]. The trailer (tr) region (155-neucleotide long) at the 3′ end of RSV antigenome has a promoter, which allows RdRp towards RSV genome synthesis [142, 143, 150]. The first 12 nucleotides of tr promoter are like those of the le promoter and the signal starts from position +1 and + 3 undergoes replication and transcription, respectively, but tr promoter cannot produce capped and polyadenylated mRNA because of lacking ge signal sequence adjacent to tr promoter [151, 152]. However, it is reported that tr promoter can initiate transcription of short RNA, which can inhibit cellular stress granules [153]. The concentration of ATP or GTP can determine the fate of replication and transcription at positions +1 (1 U) or position +3 (3C) observed at in vitro model, specifically, higher ATP concentration stimulates initiation from 1 U and evades initiation at 3C, in contrast, higher GTP concentration displays opposite effect [154]. Overall, L and P proteins form the core RdRp and L-P complex then form L-P-N and L-P-M2–1 complex to initiate replication and transcription, respectively [79, 155].

5.3 Virion assembly and budding

Both assembly and budding of RSV occur at the apical side of ciliated cells [156]. RSV assembly is associated with lipid microdomain or lipid raft rich in cholesterol and sphingolipids; specifically, RSV filament formation observed in caveolin-1 and lipid-raft ganglioside GM1 rich regions of host cell surface membrane [157, 158, 159]. RSV assembly into viral filament occurs at the cell surface requiring the activity of F protein cytoplasmic tail and M protein and this process are not dependent on actin polymerization [160]. However, Mehedi et al., showed the depletion of ARP2 resulted in perturbation of RSV progeny virion on the infected cell surface, consequently reducing viral shedding [8]. Viral assembly requires the activity of F protein cytoplasmic tail and M protein because both proteins accumulate in inclusion bodies cytoplasmic tail of F protein enables the release of the complex of matrix and RNP from inclusion bodies [161]. Although previous studies showed that three proteins including M, P, and F proteins are enough to create virus-like particles, a recent nuclear magnetic resonance study suggests that three novel interaction sites of M on P including site I in αN2 region, site II in 115 to 125 region and oligomerization domain where oligomerization domain is necessary for virus-like structure formation and virus release [137]. The incorporation of RSV proteins into lipid microdomains during virus assembly can cause the interaction of F protein with host factors including caveolin-1, CD44, RhoA, causing microvillus-like projections essential for virus filament and syncytium formation [162, 163]. Actin cytoskeleton and actin-associated pathways linked with PI3K and Rac GTPase are involved in RSV assembly [164]. M protein can bind DNA as well as RNA and it localizes into the nucleus mediated by importin-β1 nuclear import receptor, which forms a complex with guanine nucleotide-binding protein Ran and binds M protein amino acid 100–183 [165, 166]. During the early phase of infection, nuclear accumulation of M protein was observed when M protein interacts with nuclear components mediated by its zinc finger domain resulting in the inhibition of host cell transcription [165]. During the later phase of infection, M protein undergoes phosphorylation inducing nuclear export mediated by Crm1 by unmasking the nuclear export signal [78]. Therefore, M protein is thought to play a regulatory role as a transcription inhibitory factor by inhibiting viral transcriptase to facilitate RSV assembly and budding [77, 167]. RSV glycoprotein and RNP vesicles combined together prior to the filamentous virus formation and G protein recycling has been observed via clathrin-mediated endocytosis, which might be connected with filamentous RSV formation [168]. RSV budding preferentially appears at the apical membrane of epithelial cells by an apical recycling endosome (ARE)-mediated apical protein sorting pathway [169]. RSV budding is independent of the endosomal sorting complex necessary for transport (ESCRT) mechanism controlled by ARE-associated protein, Rab11 family interacting protein 2 (FIP2) [170]. Recently, ARP2 is identified as a novel host factor of RSV budding and cell-to-cell spread [8].

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6. RSV cell-to-cell spread

Although RSV progeny virions mostly remain cell-associated, virus shedding occurs from the infected cell’s surface and through cellular protrusions namely filopodia [8, 9]. RSV-induced syncytium (multinucleated cell) formation is a common feature of RSV infection in vitro. The syncytium involves the merging of infected cells with the adjacent uninfected cells, which allows the transfer of viral components from infected cells to the adjacent uninfected cells [171] (Figure 3). Mehedi et al., first showed that RSV uses a novel filopodia-driven cell-to-cell spread mechanism in the lung epithelial cells in vitro (Figure 4). It appears that RSV infection modulates cellular actin dynamics; particularly, actin-related protein 2/3 (ARP2/3) complex-driven actin polymerization contributes to lamellipodium and filopodium formation of cell motility. They showed the depletion of ARP2, a major constituent of the ARP2/3 complex resulted in a substantial reduction of RSV budding and filopodia-driven cell-to-cell spread [8, 172, 173, 174].

Figure 3.

RSV-induced syncytium (multinucleated cell) formation. A549 cells were infected with GFP-expressing RSV (RSV-GFP) at a multiplicity of infection of 1. At 48-hour post-infection, cells were fixed and imaged under an epifluorescence.

Figure 4.

Filopodia-driven RSV cell-to-cell spread. A549 cells were infected with RSV-WT (strain A) at a multiplicity of infection of 1. At 24-hour post-infection, cells were fixed, permeabilized, and stained for RSV F protein by using F-specific immunofluorescence (IFA) (green). F-actin was detected by rhodamine phalloidin staining (red). The image was taken under a stimulated emission depletion (STED) microscope.

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Written By

Sattya Narayan Talukdar and Masfique Mehedi

Submitted: 24 February 2022 Reviewed: 01 April 2022 Published: 28 July 2022

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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