Mutants Of Respiratory Syncytial Virus Research Articles

Respiratory Syncytial Virus Matrix Protein Is Sufficient and Necessary to Remodel Host Mitochondria in Infection.

Although respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants, immunosuppressed adults and the elderly worldwide, there is no licensed RSV vaccine or widely applicable antiviral therapeutics We previously reported a staged redistribution of mitochondria with compromised respiratory activities and increased reactive oxygen species (ROS) generation during RSV infection. Here, we show for the first time that the RSV matrix protein (M) is sufficient and necessary to induce these effects. Ectopically expressed M, but not other RSV proteins, was able to induce mitochondrial perinuclear clustering, inhibition of mitochondrial respiration, loss of mitochondrial membrane potential (Δψm), and enhanced generation of mitochondrial ROS (mtROS) in infection. Truncation and mutagenic analysis revealed that the central nucleic acid-binding domain of M is essential for the effects on host mitochondria, with arginine/lysine residues 170/172 being critically important. Recombinant RSV carrying the arginine/lysine mutations in M was unable to elicit effects on host mitochondria. Further, wild-type but not mutant RSV was found to inhibit the mRNA expression of genes encoding mitochondrial proteins, including Complex I subunits. Importantly, the RSV mutant was impaired in virus production, underlining the importance of M-dependent effects on mitochondria to RSV infection. Together, our results highlight M's unique ability to remodel host cell mitochondria and its critical role in RSV infection, representing a novel, potential target for future anti-RSV strategies.

Viral RNA N6-methyladenosine modification modulates both innate and adaptive immune responses of human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N6-methyladenosine (m6A) and RSV grown in m6A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m6A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m6A methylation enhances innate immune responses which in turn promote adaptive immunity.

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis.IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.

Discovery of a potent dual inhibitor of wild-type and mutant respiratory syncytial virus fusion proteins through the modulation of atropisomer interconversion properties

The development of effective respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors against both wild-type and the D486N-mutant F protein is urgently required. We recently reported a 15-membered macrocyclic pyrazolo[1,5-a]pyrimidine derivative 4 that exhibited potent anti-RSV activities against not only wild-type, but also D486N-mutant F protein. However, NMR studies revealed that the 15-membered derivative 4 existed as a mixture of atropisomers. An optimization study of the linker moiety between the 2-position of the benzoyl moiety and the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold identified a 16-membered derivative 42c with an amide linker that showed a rapid interconversion of atropisomers. Subsequent optimization of the 5-position of the pyrazolo[1,5-a]pyrimidine scaffold and the 5-position of the benzoyl moiety resulted in the discovery of a potent clinical candidate 60b for the treatment of RSV infections.

Discovery of a Potent Dual Inhibitor of Wild-Type and Mutant Respiratory Syncytial Virus Fusion Proteins.

A novel series of macrocyclic pyrazolo[1,5-a]pyrimidine derivatives as respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors were designed and synthesized based on docking studies of acyclic inhibitors. This effort resulted in the discovery of several macrocyclic compounds, such as 12b, 12f, and 12h, with low nanomolar to subnanomolar activities against the wild-type RSV F protein A2. In addition, 12h showed a single-digit nanomolar potency against the previously reported drug-resistant mutant D486N. Molecular modeling and computational analyses suggested that 12h binds to the D486N mutant while maintaining a rigid bioactive conformation via macrocyclization and that it interacts with a hydrophobic cavity of the mutant using a new interaction surface of 12h. This report describes the rational design of macrocyclic compounds with dual inhibitory activities against wild-type and mutant RSV F proteins.

Dichloro-Phenyl-Benzotriazoles: A New Selective Class of Human Respiratory Syncytial Virus Entry Inhibitors.

Human Respiratory Syncytial Virus (RSV) is the primary cause of bronchopneumonia in infants and children worldwide. Clinical studies have shown that early treatments of RSV patients with ribavirin improve prognosis, even if the use of this drug is limited due to myelosuppression and toxicity effects. Furthermore, effective vaccines to prevent RSV infection are currently unavailable. Thus, the development of highly effective and specific antiviral drugs for pre-exposure prophylaxis and/or treatment of RSV infections is a compelling need. In the quest of new RSV inhibitors, in this work we evaluated the antiviral activity of a series of variously substituted 5,6-dichloro-1-phenyl-1(2)H-benzo[d][1,2,3]triazole derivatives in cell-based assays. Several 1- and 2-phenyl-benzotriazoles resulted fairly potent (μM concentrations) inhibitors of RSV infection in plaque reduction assays, accompanied by low cytotoxicity in human highly dividing T lymphoid-derived cells and primary cell lines. Contextually, no inhibitory effects were observed against other RNA or DNA viruses assayed, suggesting specific activity against RSV. Further results revealed that the lead compound 10d was active during the early phase of the RSV infection cycle. To understand whether 10d interfered with virus attachment to target cells or virus-cell fusion events, inhibitory activity tests against the RSV mutant strain B1 cp-52—expressing only the F envelope glycoprotein—and a plasmid-based reporter assay that quantifies the bioactivity of viral entry were also performed. The overall biological results, in conjunction with in silico modeling studies, supported the conclusion that the RSV fusion process could be the target of this new series of compounds.

Molecular Requirements for Self-Interaction of the Respiratory Syncytial Virus Matrix Protein in Living Mammalian Cells.

Respiratory syncytial virus (RSV) is an important human pathogen, which infects respiratory tract epithelial cells causing bronchiolitis and pneumonia in children and the elderly. Recent studies have linked RSV matrix (M) ability to self-interaction and viral budding. However, RSV M has been crystalized both as a monomer and a dimer, and no formal proof exists to date that it forms dimers in cells. Here, by using a combination of confocal laser scanning microscopy and bioluminescent resonant energy transfer applied to differently tagged deletion mutants of RSV M, we show that the protein can self-interact in living mammalian cells and that both the N and C-terminus of the protein are strictly required for the process, consistent with the reported dimeric crystal structure.

Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR-29a-mediated inhibition of interferon-α receptor

Human respiratory syncytial virus (RSV) non-structural protein 1 (NS1) has recently been suggested to inhibit type-I interferon (IFN)-dependent immune responses during RSV infection. However, the precise function of RSV NS1 protein in reducing the antiviral effects of IFNs against RSV is poorly understood. The roles of cellular miRNAs in the defence against RSV infection are not well characterized. In this study, qRT-PCR analysis revealed that miR-29a expression was upregulated in the recombinant wild-type RSV (rRSV-WT) group compared with the control group, but no changes were observed in the recombinant RSV mutant lacking NS1 (rRSV-ΔNS1) group. Using dual-luciferase reporter assay, we demonstrated that miR-29a could directly target IFNAR1 3′-UTR and downregulate IFNAR1 expression. In addition, RSV NS1 suppressed IFNAR1 expression at both RNA level and protein level in human lung adenocarcinoma cell line A549. RSV plaque assays showed that the number of RSV plaques in miR-29a mimics group was significantly higher than that in miR-29a inhibitor group or miRNA scramble control group. HA-NS1 overexpression increased the numbers of RSV plaque, but the promotive effect on virus replication was attenuated in cells transfected with miR-29a inhibitors. These results suggest that miR-29a, upregulated during RSV infection, is a negative regulator of IFNAR1 and is critical for RSV NS1-induced virus replication.

Reverse Genetics of Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a negative-strand RNA virus that is associated with severe lower respiratory tract infections in young infants and the elderly. RSV remains a leading cause worldwide of infant mortality, and despite the high clinical and economic burden of the virus there are currently no available vaccines. Here, we describe the methods for recovery of recombinant RSV viruses using a bacterial artificial chromosome and methods related to procurement and expansion of stocks of RSV mutants.

Murine immune responses to virus-like particle-associated pre- and postfusion forms of the respiratory syncytial virus F protein.

Virus-like particles (VLPs) built on the Newcastle disease virus (NDV) core proteins, NP and M, and containing two chimeric proteins, F/F and H/G, composed of respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective RSV neutralizing antibodies in mice. Here, we report the properties of VLPs constructed to contain mutant RSV F protein ectodomains stabilized in prefusion (pre-F/F) or postfusion (post-F/F) configurations. The structures of the chimeric proteins assembled into VLPs were verified immunologically by their reactivities with a conformationally restricted anti-F protein monoclonal antibody. Following immunization of mice, without adjuvant, pre-F/F-containing VLPs induced significantly higher neutralizing antibody titers than the post-F/F-containing VLPs or the wild-type F/F-containing VLPs after a single immunization but not after prime and boost immunization. The specificities of anti-F IgG induced by the two mutant VLPs were assessed by enzyme-linked immunosorbent assay (ELISA) using soluble forms of the prefusion and postfusion forms of the F protein as targets. While both types of VLPs stimulated similar levels of IgG specific for the soluble postfusion F protein, titers of IgG specific for prefusion F induced by the pre-F/F-containing VLPs were higher than those induced by post-F/F-containing VLPs. Thus, VLPs containing a stabilized prefusion form of the RSV F protein represent a promising RSV vaccine candidate. The development of vaccines for respiratory syncytial virus has been hampered by a lack of understanding of the requirements for eliciting high titers of neutralizing antibodies. The results of this study suggest that particle-associated RSV F protein containing mutations that stabilize the structure in a prefusion conformation may stimulate higher titers of protective antibodies than particles containing F protein in a wild-type or postfusion conformation. These findings indicate that the prefusion F protein assembled into VLPs has the potential to produce a successful RSV vaccine candidate.

The Thr205 Phosphorylation Site within Respiratory Syncytial Virus Matrix (M) Protein Modulates M Oligomerization and Virus Production

Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.

Respiratory syncytial virus and palivizumab: Where are we in 2014?

Respiratory syncytial virus and palivizumab: Where are we in 2014?

Major Histocompatibility Complex-Dependent Cytotoxic T Lymphocyte Repertoire and Functional Avidity Contribute to Strain-Specific Disease Susceptibility after Murine Respiratory Syncytial Virus Infection

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.

Identification of Deletion Mutant Respiratory Syncytial Virus Strains Lacking Most of the G Protein in Immunocompromised Children with Pneumonia in South Africa

Respiratory syncytial virus (RSV) G protein deletion mutants replicate effectively in vitro but have not been detected in nature. Subtyping of RSV strains in hospitalized children in South Africa identified G protein PCR amplicons significantly reduced in size in 2 out of 209 clinical specimens screened over 4 years. Sequence analysis revealed subtype B strains lacking nearly the entire G protein ectodomain in one HIV-positive and one HIV-exposed child hospitalized with pneumonia. The association of clinical strains lacking most of the G protein with lower respiratory tract infection in immunocompromised children may have implications for RSV vaccine development.

Respiratory Syncytial Virus Proteins Modulate Suppressors of Cytokine Signaling 1 and 3 and the Type I Interferon Response to Infection by a Toll-Like Receptor Pathway

Respiratory syncytial virus (RSV) is a common cause of repeat infections throughout life and potentially severe lower respiratory tract illness in infants, young children, and the elderly. RSV proteins have been shown to contribute to immune evasion by several means, including modification of cytokine and chemokine responses whose expression is negatively regulated by suppressor of cytokine signaling (SOCS) proteins. In this study, we examine the role of SOCS1 and SOCS3 regulation of the type I interferon (IFN) response in normal fully-differentiated human bronchial epithelial cells infected with RSV or with an RSV mutant virus lacking the G gene. The results show that RSV G protein modulates SOCS expression to inhibit type I IFN and interferon-stimulated gene (ISG)-15 expression very early as well as late in infection, and that SOCS induction is linked to toll-like receptor (TLR) signaling by RSV F protein, as indicated by interferon-regulatory factor (IRF)-3 activation and nuclear translocation. These findings indicate that RSV surface proteins signal through the TLR pathway, suggesting that this may be an important mechanism to reduce type I IFN expression to aid virus replication.

Nonstructural Proteins of Respiratory Syncytial Virus Suppress Premature Apoptosis by an NF-κB-Dependent, Interferon-Independent Mechanism and Facilitate Virus Growth

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-kappaB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.

In vivo selection of respiratory syncytial viruses resistant to palivizumab.

Palivizumab (PZ) is the only monoclonal antibody currently available for use in humans against an infectious disease. PZ is administered prophylactically for respiratory syncytial virus (RSV) infections. RSV selected in cell culture for growth in the presence of PZ develops F gene mutations and can be resistant to PZ prophylaxis in cotton rats. Here, we evaluated the potential for PZ-resistant RSV mutants to arise in vivo. Cotton rats were immunosuppressed with cyclophosphamide, administered PZ, and inoculated intranasally with RSV. Lungs were harvested 12 weeks after RSV infection, reverse transcription-PCR-amplified F gene fragments were cloned into plasmids, and the nucleotide sequences of the cloned cDNAs were determined. Three of the five animals had mixed populations of lung virus, and over 50% of the clones from the three animals revealed F gene mutations associated with resistance to PZ. A virus completely resistant to PZ neutralization was recovered from the lung homogenate of a rat that had received PZ. Thus, prolonged pulmonary replication of RSV in the presence of PZ was followed by the appearance of viruses resistant to PZ. The potential for the development of resistance is a consideration as the antibody is used prophylactically against RSV and as passively administered antibodies are under development for other infections, including emerging viruses and agents of biodefense.

Variable Resistance to Palivizumab in Cotton Rats by Respiratory Syncytial Virus Mutants

Palivizumab (PZ) is the only monoclonal antibody in use against a human infectious disease. PZ is given as prophylaxis against infection with respiratory syncytial virus (RSV). An RSV escape mutant, MP4, has been shown to resist PZ prophylaxis in cotton rats. To further define the potential of RSV to resist prophylaxis, additional PZ-resistant viruses were selected in cell culture and were tested for susceptibility to PZ in cotton rats. Mutant MS412 had an A-->C mutation at nucleotide position 827 in the F gene, resulting in an amino acid change from Lys to Gln at position 272. Mutant F212 had an A-->T mutation at position 816, leading to an amino acid change from Asn to Ile at position 268. In vitro, F212 had impaired growth kinetics. In cell culture, F212 was partially and MS412 was completely resistant to PZ neutralization. A single prophylactic dose of 15 mg/kg PZ protected cotton rats from infection with F212 but not with MS412. Both in vitro and in vivo, individual RSV PZ escape mutants varied in their susceptibility to PZ. Mutations associated with resistance to PZ did not always result in failure of PZ prophylaxis.

Recombinant respiratory syncytial virus lacking secreted glycoprotein G is attenuated, non-pathogenic but induces protective immunity

Respiratory syncytial virus (RSV) causes intense pulmonary inflammatory responses in some infected infants. The surface attachment protein ‘G’ of RSV has membrane-bound and secreted forms and shows homology to the CX3C chemokine fractalkine. Using recombinant techniques, we generated replication-competent recombinant clonal RSV expressing normal G proteins (‘rRSV’) or only the membrane-bound form of G (‘Gmem rRSV’). Both recombinants grew well in HEp-2 cells, but after primary intranasal infection in mice, pulmonary Gmem rRSV replication was reduced tenfold compared to parental or rRSV; moreover, CCL2 and CCL5 production was greatly reduced and no apparent disease or pulmonary cellular infiltration was observed. However, Gmem rRSV-infected mice developed good antibody responses and were fully protected against subsequent intranasal challenge with parental virus. Even in mice sensitized to G by cutaneous infection with recombinant vaccinia expressing G, intranasal challenge with Gmem rRSV caused insignificant disease. We conclude that secreted G is a key viral product assisting virus replication in vivo, enhancing CCL2 and CCL5 production and promoting illness. Engineered RSV mutants lacking the ability to secrete G are thus promising vaccine candidates.

Inhibition of respiratory syncytial virus infection with the CC chemokine RANTES (CCL5).

Respiratory syncytial virus (RSV) is a major cause of respiratory tract disease in infants, aged adults, and immunosuppressed patients. The only approved medicines for RSV disease are administration of prophylatic antibodies or treatment with a synthetic nucleoside. Both approaches are expensive and the latter is not without risk and of controversial benefit. The present investigation studied whether pharmaceutical or biologic compounds based upon chemokines might be useful in preventing RSV disease. Of interest was RANTES/CCL5, which inhibits infection by HIV strains that use chemokine receptor (CCR)-5 as co-receptor. Herein, we report that prior or simultaneous treatment of HEp-2 cells with recombinant human CCL5 provides dose-dependent inhibition of infection with RSV. Other recombinant chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-2/CCL8, eotaxin/CCL11, MIP-1delta/CCL15, stromal cell derived factor (SDF)-1alpha/CXCL12) were not inhibitory. The data suggested that CCL5 might inhibit infection by blocking fusion (F) protein-epithelial cell interactions. Infections by mutant RSV strains deleted of small hydrophobic and/or attachment proteins and only expressing F protein in the envelope were inhibited by prior treatment with CCL5 or a biologically inactive N-terminally modified met-CCL5. Inhibition was also observed when virus adsorption and treatment with CCL5 were performed at 4 degrees C. Flow cytometry further revealed that epithelial cells were positive for CCR3, but not CCR1 or CCR5. Thus, novel mimetics of CCL5 may be useful prophylatic agents to prevent respiratory tract disease caused by RSV.