Differentiation of Th Subsets Inhibited by Nonstructural Proteins of Respiratory Syncytial Virus Is Mediated by Ubiquitination

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, constitutes an important risk factor for the development of subsequent asthma. However, the mechanism underlying RSV-induced asthma is poorly understood. Viral non-structural proteins NS1 and NS2 are critically required for RSV virulence; they strongly suppress IFN-mediated innate immunity of the host cells. In order to understand the effects of NS1 and NS2 on differentiation of Th subsets, we constructed lentiviral vectors of NS1 or NS2 to infect 16 HBE and analyzed the expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 by Flow Cytometric Analysis and real-time PCR. The results showed that NS1 inhibited expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 lymphocytes, which could be reversed by deleting elongin C binding domain. NS2 inhibited the differentiation of Th2 and Th17, which was reversed by proteasome inhibitors of PS-341. Our results indicated that NS1 inhibited the differentiation of T lymphocytes through its mono-ubiquitination to interacted proteins, while NS2 inhibited differentiation of Th2 and Th17 through ubiquitin-proteasome pathway, which may be related with the susceptibility to asthma after RSV infection.     

Respiratory Syncytial Virus Infection of the Newborn

In an outbreak of respiratory syncytial (R.S.) virus infection in a maternity hospital the respiratory illness was of a mild nature and the virus was not found in infants without respiratory symptoms. This confirms the suggestion that R.S. virus can infect infants at a very early age. Rapid diagnosis was achieved by applying the direct fluorescent antibody technique to cells in nasal secretions. This proved to be more sensitive than culture techniques where there was delay between the onset of respiratory symptoms and submission of specimens to the laboratory.     

Temperature-sensitive Mutants of Respiratory Syncytial Virus

Four conditional-lethal temperature-sensitive mutants of RS virus were detected among the progeny of 454 plaques derived from virus grown in the presence of 10(-4)m 5-fluorouridine. These mutants were stable (reversion frequency, 10(-5.0) or less and failed to produce plaques at 38 or 39 C. Plaquing efficiency was depressed 100-fold or more at 37 C. Variable suppression of growth at the restrictive temperature of 39 C was observed, ranging from 16-fold to complete suppression. The temperature-sensitive defect of three of the mutants appeared to affect functions which were expressed late in the replicative cycle. One of the mutants produced atypical nonsyncytial plaques.     

The Respiratory Syncytial Virus Polymerase Has Multiple RNA Synthesis Activities at the Promoter

Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1–25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3′ end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3′ end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3′ terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.     

Affinity-Purified Respiratory Syncytial Virus Antibodies from Intravenous Immunoglobulin Exert Potent Antibody-Dependent Cellular Cytotoxicity

Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections.     

Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.     

Morphogenesis and Ultrastructure of Respiratory Syncytial Virus

Respiratory syncytial (RS) virus was grown in Vero cells and fixed for electron microscopy at various stages of maturation. Both filamentous and round or kidney-shaped particles, either with (complete) or without (incomplete) internal structure, were observed. All four morphological forms were identical with respect to their reactivity with ferritin-labeled antibody to RS virus. Freezeetching revealed a structural feature apparently unique for RS virus, namely helical striations around the core on the internal aspect of the envelope. This specific configuration was already detectable during the early stages of viral differentiation of the host cell membrane. Concentration of free virus by zonal ultracentrifugation of culture fluids onto sucrose cushions yielded predominantly filamentous forms up to 10 mum in length.     

婴幼儿呼吸道合胞病毒感染的治疗

呼吸道合胞病毒(respiratory syncytial virus RSV)是引起婴幼儿毛细支气管炎最重要的病原体。其主要临床表现为喘息。部分患儿可出现反复喘息发作而发展为哮喘。对RSV感染尚无特效治疗药物,仍然以支持对症治疗为主,目前的研究热点是中西医结合治疗,反义基因治疗,高渗盐水雾化吸入治疗等。     

Structural and Nonstructural Proteins of Saint Louis Encephalitis Virus

Analysis of purified Saint Louis encephalitis (SLE) virus by acrylamide gel electrophoresis revealed that the virions contained three structural proteins designated SP-1, SP-2, and SP-3 which had molecular weights of 63,000, 18,000, and 8,500, respectively. The envelope contained proteins SP-1 and SP-3 which were removed from the nucleocapsid by nonionic detergent treatment. Nucleocapsids prepared by deoxycholate treatment of complete virions had a density of 1.301 in potassium tartrate and contained SP-2 and SP-3. Brij-58-prepared SLE nucleocapsids had a density of 1.321 and contained only SP-2. Cycloheximide treatment for 1 hr in the presence of actinomycin irreversibly inhibited BHK cellular protein synthesis and reversibly inhibited the synthesis of SLE viral protein and ribonucleic acid. Three structural proteins and five virus-specific nonstructural proteins were detectable in SLE virus-infected BHK cells treated with actinomycin and pulse-inhibited with cycloheximide. Formation of each individual viral structural protein was detectable within 30 min after cycloheximide removal and continued with only minor changes from 12 to 18 hr after infection. Late in the infection cycle, synthesis of the nucleocapsid structural protein SP-2 and SP-3, the small envelope protein, was no longer detectable.     

Prospects For the Use of Peptides against Respiratory Syncytial Virus

Molecular Biology - The human respiratory syncytial virus (RSV) is one of the most common viral pathogens that affects the lower respiratory tract and could be a reason of bronchiolitis and/or...     

Intrapatient Variation of the Respiratory Syncytial Virus Attachment Protein Gene

Intrapatient variability of the attachment (G) protein gene of respiratory syncytial virus (RSV) was examined using both population and single-genome sequencing. Samples from three patients infected with a group B virus variant which has a 60-nucleotide duplication in the G protein gene were examined. These samples were chosen because occasional mixed sequence bases were observed. In a minority of RSV genomes from these patients considerable variability was found, including point mutations, insertions, and deletions. Of particular note, the deletion of the exact portion of the gene which had been duplicated in some isolates was observed in viral RNAs from two patients.Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and vulnerable adults (3, 9) and is unusual in that it can repeatedly reinfect individuals (5, 6). RSV isolates are classified into two groups, A and B, and the attachment (G) protein, a target for neutralizing antibodies, is the most variable of the viral proteins, showing considerable genetic and antigenic variability both within and between the groups (7, 8). The G protein is able to accommodate drastic changes, which have been observed both in culture during the selection of monoclonal antibody escape mutants (4, 12, 13) and in vivo with the emergence of new variants, including a group B strain with a duplication of 60 nucleotides (17). This strain with a 60-nucleotide duplication was first reported from Buenos Aires in 1999 (17) and then was subsequently detected in samples from 1998 in Madrid (16). The strain then became the dominant group B strain worldwide, indicating a selective advantage for this variant (16, 18). Thus, major genetic changes can be introduced into the G gene sequence while the virus replicates in its natural host, which can then be selected under favorable epidemiological conditions.Previous investigations of the genetic diversity of RSV exploited direct sequencing of PCR-amplified products (2), which represent the population average of the in vivo variants. Such sequences are derived from multiple copies of cDNA and represent the dominant sequence, and they thus do not allow detection of minority populations below about 20% prevalence (11). Information on intrapatient viral diversity during infections may therefore be missed, knowledge of which could be important in the overall understanding of the genetic diversity of this virus. We report here the analysis of individual RSV RNA molecules derived by single-genome amplification (SGA) and sequencing from clinical samples using a methodology developed for the analysis of HIV genomes (11, 14).RSV-positive samples were collected from infants admitted to Kilifi District Hospital, Kenya (10). Viral RNA extraction and cDNA synthesis were carried out as previously described (15). For population sequencing, a nested PCR was carried out on the cDNA using primers that amplified the ectodomain-coding part of the G protein gene, with the PCR product being directly sequenced. In the 2007-2008 RSV epidemic in Kilifi, group B viruses were predominant. By population sequencing of ∼100 group B samples, all were found to have the 60-nucleotide duplication observed in the Buenos Aires variant (data not shown). However, in some specimens there were some mixed bases at some positions, so the variability at the level of the single cDNA molecule was further investigated.Three samples that gave occasional mixed signals in the sequence chromatograms were further analyzed by SGA and sequencing. For SGA the cDNAs were serially diluted 3-fold up to 1:6,361. Ten nested PCRs were carried out on each dilution using Platinum high-fidelity PCR Supermix (Invitrogen) (containing Taq polymerase together with the proofreading enzyme Pyrococcus species GB-D polymerase). Based on the Poisson distribution, it has been shown that for a sample dilution yielding approximately 30% positive PCRs there is an 80% likelihood that each PCR is derived from a single cDNA molecule (11). For each of the identified endpoint dilutions, the cDNA was amplified in 80 separate nested PCRs using the high-fidelity enzyme and the positive reaction products sequenced. The nomenclature for the sequences reported in this paper is place of isolation (Kenya [Ken])/year of isolation/strain number. For SGA sequences an additional Roman number is given.The predicted length derived by population sequencing of the G proteins of the three samples examined by SGA was 310 amino acids, showing a 6-nucleotide deletion and a changed stop codon relative to the Buenos Aires strain (Fig. ​(Fig.1).1). The dominant sequences represented 60 to 88% of the sequences derived by SGA. The differences were due to point mutations, duplications, and deletions, as summarized in Table ​Table1;1; the consequences of these changes for the predicted length of the G protein are shown in Table ​Table22.Open in a separate windowFIG. 1.Nucleotide sequence alignment of part of the G protein gene (from nucleotide 400) of the sample 2 population sequence (Ken/08/80900) and a minority sequence (Ken/08/80900/ii), with the sequences of prototype group B strain CH18537 (accession number {"type":"entrez-nucleotide","attrs":{"text":"M17213","term_id":"333942","term_text":"M17213"}}M17213) and Buenos Aires strain BA/3833/99B (accession number {"type":"entrez-nucleotide","attrs":{"text":"AY333362","term_id":"33355616","term_text":"AY333362"}}AY333362). This shows the duplication of 60 nucleotides in the Kenyan and Buenos Aires viruses relative to CH18537 and the loss of the same 60 nucleotides in the Kenyan minority sequence. Termination codons are underlined.

TABLE 1.

Diversity in the SGA-derived sequences