Respiratory syncytial virus (RSV), a member of the Pneumovirus genus within the family Paramyxoviridae, is the single most important viral respiratory pathogen infecting infants and young children worldwide, as well as an important cause of respiratory tract illness in the elderly, transplant patients, and immune suppressed [12, 22, 33, 48, 51]. The RSV genome (15 kb) is single-stranded, negative-sense RNA that contains 10 transcription units which are sequentially transcribed to produce 11 proteins in the following order: NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L . The NS1 and NS2 non-structural proteins are not expressed on the virion but are two of the most abundantly expressed RNAs in RSV-infected cells due to their promoter-proximal location [5, 11, 15] These accessory proteins have been shown to act cooperatively to suppress the activation and nuclear translocation of the IFN-regulatory factor IRF-3 [4, 47], and inhibit the type I IFN signaling cascade by mediating proteosome degradation of signal transducer and activator of transcription 2 (STAT2) with Elongin-Cullin E3 ligase [10, 29].
Additionally, constructs of "humanized" NS1 and NS2 recombinant protein expressed in Escherichia coli have been shown to decrease STAT2 levels as well as type I IFN responsiveness , and recent RNA interference (RNAi) studies in mice targeting NS proteins for silencing by short interfering RNA (siRNA) resulted in inhibition of RSV replication in mice . The NS1 and NS2 proteins may also function to facilitate RSV replication outside the interferon arena as they have an anti-apoptotic effect on RSV-infected A549 cells thereby enhancing viral replication .
Increasing evidence suggests that other RSV proteins, particularly the surface proteins on the virion, have important roles in facilitating RSV infection and replication . The RSV surface attachment protein, i.e. G protein, has been shown to modify pulmonary trafficking of immune cells , as well as the pattern and type of cytokine and chemokine expression by bronchoalveolar leukocytes (BAL) and bronchoepithelial cells in RSV-infected mice [53, 55] and in RSV-infected humans [2, 23, 49]. The G protein has been shown to have a CX3C chemokine motif in the central conserved region of the protein that can mimic some of the activities of fractalkine, the only known CX3C chemokine, specifically binding to CX3CR1 and mediating CX3C-CX3CR1 leukocyte chemotaxis [16, 54]. Importantly, anti-G protein antibody responses after recent RSV infection or vaccination in humans are associated with inhibition of RSV G protein CX3C-CX3CR1 interaction and G protein-mediated leukocyte chemotaxis .
The G protein has also been shown to inhibit Toll-like receptor (TLR) 3/4-mediated IFN-beta induction , a feature that may facilitate virus replication. Interestingly, the RSV F protein has been shown to induce aspects of innate immunity through TLR4 signaling , and TLR4-deficient mice challenged with RSV exhibit impaired NK cell and CD14+ cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4 . In addition, TLR4 polymorphisms in humans are linked to impaired responses to respiratory syncytial virus  and the genetic predisposition to severe RSV infection . These features appear contradictory to facilitating RSV replication, but F protein activation of TLR signaling may be an important feature to desensitize TLR activation of immunity. For example, RSV has been shown to mediate long-term desensitization of lung alveolar macrophages to TLR ligands . This feature may be linked to the lack of durable protective immunity associated with RSV infection [50, 51]. Finally, the RSV SH protein is linked to altered Th1-type cytokine and chemokine expression by BAL cells , and can inhibit TNFα signaling . Taken together, RSV surface proteins have immune modulatory features that appear to facilitate infection and replication.
It is not surprising that TLRs have an important role in the host response to RSV infection. Viral infection has been shown to activate TLRs and retinoic acid inducible gene I (RIG-I) signaling pathways leading to phosphorylation of interferon regulatory factor3 (IRF3) and IRF7 and stimulation of type I interferon (IFN) transcription, a process important for innate antiviral immunity . Production of type I IFN depends on activation of IRF3 and IRF7 [20, 35, 44] where type I IFN expression is negatively regulated by suppressor of cytokine signaling (SOCS) proteins [7, 24]. SOCS proteins are mainly regulated at the transcriptional level but can be directly induced by stimulation of TLRs where they do not interfere with direct TLR signaling, but instead regulate paracrine IFN signaling . The SOCS protein family is comprised of eight proteins (CIS, cytokine-inducible SH2-containing protein, SOCS1-7) of structural and functional homology [7, 24]. Of the family members, SOCS1 and SOCS3 appear to be the most effective in regulating type I IFN expression. SOCS1 can directly associate with high affinity to all Janus kinases (JAKs) directly inhibiting their catalytic activity, while SOCS3 functions in part by interacting with activated cytokine receptors .
Numerous studies have established that type I IFN expression regulates hundreds of host genes that include STAT1, JAK1, ERK1, MxA, RIG-I, and IRF3 [9, 14, 27, 30, 32, 68]. One important IFN-stimulated gene that encodes an ubiquitin-like protein is IFN-stimulated gene (ISG)-15 (ISG15). ISG15 is one of the earliest ISG induced by type I IFN and has been shown to target several components of the antiviral signaling pathway .
Virally-induced ISG15 promotes an antiviral state by subverting proteosome-mediated degradation of IRF3 in infected cells . As for type I IFNs, viruses have adapted to circumvent the antiviral effects of ISG15. One example is the ability of the NS1 protein of the influenza B virus to inhibit conjugation of ISG15 to target proteins . Since IFN genes are generally transcriptionally silent until induced, for example by binding of TLR-activated transcription factors to their promoters, ISG15 expression can reveal pathogen-TLR activation of the type I IFN response.
RSV infects ciliated airway epithelial cells in the respiratory tract [19, 66] and type II pneumocytes [6, 36, 58, 60, 61]. A majority of RSV studies have used the mouse model to evaluate the host response to infection. This model has been useful to understand aspects of the immunobiology of infection. Mouse lung epithelial (MLE)-15 cells offer a good option to emulate the mouse model of RSV infection as these cells are a type II pneumocyte cell line representing the distal bronchiolar and alveolar epithelium that maintain their differentiated phenotypes and functional characteristics for up to 30–40 cell culture passages . MLE-15 cells also express microvilli, SP-A, SP-B and SP-C, form basement membranes, and are capable of expressing MHC class I antigens [34, 63, 69]. In general, type II pneumocytes comprise approximately 15% of total lung cells, and are found at the air-liquid interface [37, 64]. From this position, type II pneumocyte cells are able to respond to airborne stimuli as well as interact with various immune cells such as CD8+ T cells which are known to be important immune mediators of respiratory viral infections.
The studies reported here focus on the early antiviral host response in MLE-15 cells to RSV infection and the role of RSV surface proteins in modulating this response. The studies center on SOCS1 and SOCS3 negative regulation of the type I IFN response and ISG15 expression following infection with RSV or RSV mutant viruses lacking the G gene, or NS1 and NS2 gene deletions. These results indicate an important role for SOCS1 regulation of the antiviral host response to RSV infection, and reveal a novel role for RSV G protein modulation of SOCS3, ISG15 and IFNβ mRNA expression.