Region Of Respiratory Syncytial Virus Research Articles

Potential siRNA Molecules for Nucleoprotein and M2/L Overlapping Region of Respiratory Syncytial Virus: In Silico Design

BackgroundHuman respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in the pediatric population, elderly and in immunosuppressed individuals. Respiratory syncytial virus is also responsible for bronchiolitis, pneumonia, and chronic obstructive pulmonary infections in all age groups. With this high disease burden and the lack of an effective RSV treatment and vaccine, there is a clear need for discovery and development of novel, effective and safe drugs to prevent and treat RSV disease. The most innovative approach is the use of small interfering RNAs (siRNAs) which represent a revolutionary new concept in human therapeutics. The nucleoprotein gene of RSV which is known as the most conserved gene and the M2/L mRNA, which encompass sixty-eight overlapping nucleotides, were selected as suitable targets for siRNA design.ObjectivesThe present study is aimed to design potential siRNAs for silencing nucleoprotein and an overlapping region of M2-L coding mRNAs by computational analysis.Materials and MethodsVarious computational methods (target alignment, similarity search, secondary structure prediction, and RNA interaction calculation) have been used for siRNA designing against different strains of RSV.ResultsIn this study, seven siRNA molecules were rationally designed against the nucleoprotein gene and validated using various computational methods for silencing different strains of RSV. Additionally, three effective siRNA molecules targeting the overlapping region of M2/L mRNA were designed.ConclusionsThis approach provides insight and a validated strategy for chemical synthesis of an antiviral RNA molecule which meets many sequence features for efficient silencing and treatment at the genomic level.

Antibodies to the Central Conserved Region of Respiratory Syncytial Virus (RSV) G Protein Block RSV G Protein CX3C-CX3CR1 Binding and Cross-Neutralize RSV A and B Strains

Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein-CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein-CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines.

The Insertion of Fluorescent Proteins in a Variable Region of Respiratory Syncytial Virus L Polymerase Results in Fluorescent and Functional Enzymes But with Reduced Activities

The respiratory syncytial virus (RSV) Large protein L is the catalytic subunit of the RNA-dependent RNA polymerase complex. Currently, no structural information is available for RSV L. Sequence alignments of L protein from human and bovine strains of RSV revealed the existence of two variable regions, VR1 and VR2. Following comparison with morbillivirus and rhabdovirus L genes, VR2, which is located between domains V and VI, was chosen as an insertion site for sequences encoding the epitope tag HA or the fluorescent proteins eGFP and mCherry. Recombinant tagged-L proteins co-localized with RSV N and P proteins in transfected cells. These recombinant polymerases were shown to be functional using a viral minigenome system assay, their activities being reduced by ~70% compared to the unmodified L polymerase. We have also shown by site-directed mutagenesis that the GDNQ motif (residues 810-813 for the Long strain of HRSV) is essential for L activity.

Roles of the respiratory syncytial virus trailer region: Effects of mutations on genome production and stress granule formation

The 5′ extragenic trailer region of respiratory syncytial virus (RSV) is known to be necessary for genome replication, but is more than three times the length of the 3′ leader replication promoter, raising the possibility that trailer might play an additional role in viral replication. To examine this, mutant recombinant viruses were constructed in which the trailer region was truncated or substituted with leader-complement sequence. This analysis showed that the complete trailer increased promoter activity, facilitating genome production and viral multiplication. In addition, trailer-containing viruses did not induce stress granules, whereas the leader-complement virus mutant did, resulting in poor multi-cycle viral growth. These data demonstrate that although the RSV trailer does not contain a unique essential sequence, it augments virus growth by enabling optimal genome production. In addition, a sequence at the 5′ terminal end of the trailer region allows RSV to subvert stress granule formation.

Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex.

The Respiratory Syncytial Virus (RSV) fusogenic glycoprotein F(1) was characterized using biochemical and biophysical techniques. Two heptad-repeat (HR) regions within F(1) were shown to interact. Proteinase-K digestion experiments highlight the HR1 region (located proximal to the fusion peptide sequence) of the F(1) protein to which an HR2-derived (located proximal to the membrane-spanning domain) peptide binds, thus protecting both the protein and peptide from digestion. Solution-phase analysis of HR1-derived peptides shows that these peptides adopt helical secondary structure as measured by circular dichroism. Sedimentation equilibrium studies indicate that these HR1 peptides self-associate in a monomer/trimer equilibrium with an association constant of 5.2 x 10(8) M(-2). In contrast, HR2-derived peptides form random monomers in solution. CD analysis of mixtures containing peptides from the two regions demonstrate their propensity to interact and form a very stable (T(m) = 87 degrees C), helical (86% helicity) complex comprised of three HR1 and three HR2 members.

Development of human antibodies against linear antigenic and immunogenic regions of respiratory syncytial virus (RSV) nucleocapsid and phospho-proteins shows the site-directed characteristics.

The successful development of an RSV vaccine requires a better understanding of the pathogenesis of primary infection, susceptibility to reinfection, and the immunopathology of enhanced illness in children immunized with a non-replicating RSV candidate vaccine. The exact role of different immune parameters in RSV pathogenesis remains controversial. To study the contribution of antibodies directed to the linear antigenic and immunogenic regions of the N and P proteins in the titer rise and avidity maturation of total anti-RSV antibodies. The occurrence of antibodies directed against three linear antigenic and immunogenic regions in each of the nucleocapsid (N): N3 (Thr11 to Gly30), N25 (Ser231 to Ala250) and N39 (Thr371 to Leu391), and the phospho-(P) proteins of respiratory syncytial virus (RSV), subgroup A: P49 (Pro91 to Asp110), P56 (Ser161 to Lys180) and P62 (Glu221 to Phe241), were analyzed in ELISA with (a) 32 paired sera from humans with recent or previous RSV subgroup A and/or B infection diagnosed by conventional ELISA, detection of antigen in nasopharyngeal aspirates and measurement of antibody avidity change; and (b) 40 RSV antibody-positive sera (HCS) obtained from patients during their convalescence from RSV infection and possessing clearly demonstrable titers of RSV IgG in conventional enzyme immunoassays (EIA) based on whole RSV antigen. The titer rise of antibodies directed to the combined three peptides representing the RSV N protein was well correlated with the rise in anti-RSV antibodies measured in whole antigen ELISA. Surprisingly, the rise in antibodies against a truncated main C-terminal epitope (Gln381 to Leu391) of the N protein (represented by subgroup A specific sequence of the N39/1 peptide) was inversely correlated with the titer rise of total anti-RSV antibodies. The titer rise of antibodies to the C-terminal linear site of the RSV N (N39/1) protein was subgroup-specific during the course of primary RSV infection. A titer rise in antibodies to the C-terminal linear sites of RSV N (i.e. N39/1) and P (P62) proteins had a dominating appearance in sera from newborn infants (6-7 months) and from patients with RSV reinfections. Anti-RSV antibody titers of late convalescent sera correlated with the titers of antibodies directed to the C-terminal linear site of RSV P (P62) protein. The avidity maturation of the anti-RSV immune response followed the titer rise of anti-P62 antibodies during the course of primary or secondary RSV infection.

The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome

The first nine genes of respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus, are separated by intergenic regions which range in size from 1 to 52 nucleotides for strain A2 and lack obvious consensus elements except that each ends in an A (genome sense). Their significance for gene expression was investigated by using RSV-CAT-LUC RNA, a helper-dependent cDNA-encoded dicistronic analog of RSV genomic RNA in which the viral genes were replaced by a negative-sense copy of the translational open reading frame (ORF) encoding chloramphenicol acetyltransferase (CAT) as the upstream, leader-proximal gene and that encoding luciferase (LUC) as the downstream gene. These foreign ORFs were flanked by the RSV gene-start (GS) and gene-end (GE) transcription signals and separated by the naturally occurring G/F intergenic region. The RSV-CAT-LUC minigenome was synthesized in vitro and transfected into RSV-infected cells, and synthesis of the CAT and LUC mRNAs was monitored by enzyme assay and Northern (RNA) blot hybridization. Surprisingly, substitution of each of the other naturally occurring RSV intergenic regions in turn did not significantly alter the absolute or relative amounts of the two mRNAs. Substitution of a nonnatural 10-nucleotide intergenic region, or elimination of the intergenic region altogether, also had little effect on the level of expression of the two genes. Four of the minigenome variants containing naturally occurring intergenic regions were modified further by replacing part of the LUC ORF with a second copy of the CAT ORF, so that each of the two mRNAs would hybridize equally with a CAT-specific probe and their relative molar amounts could be determined. The level of expression of the downstream gene was 0.30 to 0.36 that of the upstream one. This determined the magnitude of RSV transcriptional polarity across a gene pair and confirmed that this value was very similar among the various intergenic regions. Minigenome transcription also yielded a CAT-LUC readthrough mRNA at a level 0.10 to 0.13 that of the LUC mRNA. In summary, the structurally diverse RSV intergenic regions do not appear to play a role in modulating RSV gene expression.