*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 [63–65]. 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

*Respiratory Syncytial Virus DOI: http://dx.doi.org/10.5772/intechopen.104771*

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].
