The observed nucleotide differences apparently do not specify genetic information that may account for species specificity, further confirming the close relationship of BRSV and HRSV. Members of the genus Pneumovirus show particular features that distinguish them from other paramyxovirus genera, such as the lack of P gene RNA editing. Apparently, this is also true for BRSV. Neither the parental virus ATue nor any of the recombinants have a genome consisting of multiples of 6 nt.
The recombinant BRSV genome lacks five residues compared to the ATue genome but replicated with the same efficiency. Again, they were found to replicate at identical rates. Another peculiarity of pneumoviruses is the high number of encoded genes, some of which do not have counterparts in other paramyxoviruses, such as SH, M2, and the nonstructural genes NS1 and NS2.
A role for the M2 protein as a transcription elongation factor has also been established 5 , 16 , but the function of the two nonstructural proteins NS1 and NS2 has remained rather obscure. Only revertants of HRSV NS2 knockout viruses, in which tandem stop codons were introduced in such a way that the NS2 protein would not be expressed, could be isolated from plaques observed in transfection experiments 42 , indicating an important function of HRSV NS2 in the virus life cycle. Strikingly, in the avian pneumovirus TRTV, both nonstructural genes are absent, emphasizing the question of whether they represent essential genes in mammalian pneumoviruses such as BRSV.
The pattern and the relative amounts of mRNAs and full-length virus RNA produced in infected cells were not markedly changed, except for the lack of an NS2 transcript. Moreover, maximum titers were reached in MDBK cells infected with NS2 deletion mutants by 15 days after infection, compared to 8 days after infection with standard ATue As the cDNA constructs used for recovery of nondeficient rBRSV were made by completion of the NS2 deletion cDNA, the observed slower growth and the reduced virus titers are due to the lack of NS2 rather than to putative differences in other parts of the genome.
Thus, although not essential, NS2 is an accessory factor able to substantially support virus growth, by a thus far unknown mechanism.
Further experiments using modified virus mutants are now feasible and should help to reveal the mechanisms of NS2 involved in facilitating virus growth in different cell types. The successful recovery of the first BRSV strain from which an entire gene has been deleted is important not only for studying the molecular biology and genetics of the virus but also because it provides a severely attenuated virus with an unequivocal serological marker.
Further manipulation of NS2-deficient viruses may lead to the development of attenuated marker vaccines, easily distinguishable from wild-type virus. For the prevention of respiratory diseases, live vaccines appear to be best suited due to their ability to confer local immunity in addition to humoral immune response. It will also be of interest to determine whether foreign epitopes or proteins can be incorporated into the virion in order to design vaccines for other respiratory pathogens or vectors for transient gene therapy.
National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Ursula J. Author information Article notes Copyright and License information Disclaimer. Phone: 49 Fax: 49 E-mail: ed. Received Jul 2; Accepted Oct 8. This article has been cited by other articles in PMC.
Construction of BRSV full-length plasmids. Open in a separate window. Construction of expression plasmids pN, pP, pL, and pM2. Establishment of a cell line stably expressing phage T7 RNA polymerase. Northern hybridization of viral RNA. Nucleotide sequence accession number. Sequence of the ATue L gene. Sequence analysis of the BRSV leader and trailer regions. Identification of genetic tags and transcription analysis of recombinant virus.
Recombinant viruses lacking the NS2 gene are attenuated in cell culture. This work was supported by Intervet International B. The NS1 protein of human respiratory syncytial virus is a potent inhibitor of minigenome transcription and RNA replication. Baron M D, Barrett T. Rescue of rinderpest virus from cloned cDNA.
Bridgen A, Elliott R M. Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus. Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Rescue of synthetic helper-dependent analogs of the genomic RNAs of respiratory syncytial virus and parainfluenza virus type 3.
Vaccines modern approaches to new vaccines including prevention of AIDS. Cold Spring Harbor, N. Conzelmann K-K. Genetic manipulation of non-segmented negative-strand RNA viruses. RSV infection also results in a high number of secondary bacterial infections Heikkinen et al. Moreover, bacterial pneumonia is attributable to RSV in In contrast, bacterial pneumonia attributable to influenza infection occurs in only 3. RSV is a fascinating virus impacting large portions of the population and yet knowledge about the viral life cycle and regulation is limited.
Importantly, there is still no licensed vaccine for RSV despite the decades of attempts highlighting a need for better understanding the viral pathogenesis and mechanisms fostering immunity.
This activation pathway is summarized in Figure 1. Interferon signaling pathway altered by Ns. TLR3 pathway's arrows are in yellow. Whichever one is activated leads to slight variations in the cascade. Type 1 interferons function as antiviral defense mechanisms and as a way of alerting other cells in the vicinity of an invader. In order to undergo successful transcription, RSV requires its M protein, a transcription elongation factor, in addition to the N, P, and L proteins.
Other members of the Mononegavirales order, such as its closest relatives metapneumovirus and parainfluenza, only require the N, P and L proteins for this process. During RSV transcription, the polymerase proceeds along with the transcript from the first start site, right before the Ns1 gene, in a sequential, termination-re-initiation stop-start mechanism Kuo et al.
Since the viral genes must be transcribed in order, the order of genes in the genome is vital in the timing of infection. Ns1 and Ns2 non-structural proteins are the first genes to be transcribed. These proteins, which are essential for permissive in vivo infection, function to inhibit the type 1 interferon IFN response, other elements of the immune system, and may contribute to the high incidence of secondary bacterial infections in RSV infections due to its interference with the immune system Teng and Collins, ; Jin et al.
The non-structural genes of RSV are unique to the virus, as they have low, if any homology, with genes of other viruses in the family serving a similar function Swedan et al.
This review will further focus on the non-structural genes of RSV, the interactions of their proteins, and their importance in the viral life cycle of RSV. Of RSV's non-structural genes, Ns1 has been more studied more in-depth, and thus more of its role in infection is known than that of Ns2. In terms of immune suppression, Ns1 is the dominant player, and Ns2 may be a minor one, though important still for enhancing and supporting RSV pathogenesis. Ns1 is the first gene in the genome and follows a nt leader region thus making it the most abundant transcripts produced.
NS1 is followed directly by Ns2 with a small intergenic region of around 19nts spacing between the two open reading frames Atreya et al.
While the Ns1 protein is fairly stable after translation and can be detected in pulse chase experiments for hours, the Ns2 protein disappears rapidly in the early infection stage and is thought to have an intracellular half-life of 1hr. Both Ns proteins have been observed in multimeric forms which are thought to be linked by disulfide bridges, though the predominant form of Ns1 is thought to be its monomeric form Collins et al.
Ns1 and Ns2 can form homo- and heteromers. Ns1 traffics to the nucleus, while Ns2 as well as a Ns1-Ns2 complex traffic to the mitochondria. Most of the Ns1 and Ns2 in an infected cell resides in the mitochondria as a heteromer Swedan et al. Furter study is needed to explain why the majority of the Ns proteins reside here, though it may be a way to sequester them as a means of regulation; as Ns1 inhibits viral replication including in minigenome systems Atreya et al.
While being sequestered in the mitochondria may appear counterintuitive, the Ns1 protein is subject to post-translational modification, which may indicate an additional regulatory mechanism Atreya et al. This modification occurs by proteolytic cleavage, producing a It is unknown what function the modification has and may be a byproduct of Ns1.
As the intensity of the Ns1 band in pulse-chase experiments decreased the While Ns1 is thought to be relatively stable, the discrepancies in different papers may indicate that only certain forms of the protein are stable Huang et al.
The Ns genes inhibit the type 1 interferon response as well, so this may allow for a pause in viral replication until IFN production is inhibited and RSV can replicate without being suppressed by the innate antiviral response. RSV has an unusual replication growth curve, with slower and less productive replication and gene expression compared to other viruses of the same genome type Atreya et al.
The inhibition of replication by Ns1 may help to explain this phenomenon, especially since it is the earliest and most abundant viral protein. RSV has considerable variation in its genome between strains, and the amount of variation varies by the gene. These amino acid changes, when they occur, do appear to occur within predicted MHCI binding peptides.
Some s strains also have another change in another predicted MHCI epitope with a change from R to D at the 8th amino acid position. Going back to the sequence differences between A and B strains, most of the amino acid changes fall within predicted MHCI binding epitopes suggesting immune pressure between the A and B strains but not as much within each strain inside the same decade of circulation i.
These suggest a general lack of immune pressure on this protein as usually only one predicted CD8 T cell epitope changes but not necessary from 1 year to the following year.
Thus, the amount of sequence conservation across strains may have relevance for possible drug therapies or vaccine targets, but may also have relevance for host range determination. The Ns genes may determine host range, as human RSV is capable of infecting bovine cells and vice versa though they are not capable of replicating in cows. When a virus containing the Ns genes of a non-native host i. The Ns proteins have multiple recorded interactions with cellular and other viral proteins. Ns1 has more recorded interactions and an enormous scope of impact when compared to Ns2.
Ns1 is able to interact with Ns2, the RSV M protein, and is capable of inhibiting multiple parts of the immune system Evans et al. Ns2, however, seems to be more limited in its inhibitory capacity as most data on its interaction record it only inhibiting the interferon response in infected cells.
Together the Ns genes are capable of interfering with both the adaptive and innate antiviral immune response pathways Atreya et al. The Ns proteins are known to bind to and inhibit various molecules in the type 1 interferon response signal cascade of either the RIG-I or TLR pathways. Ns1 and Ns2 form degradosomes that home to the mitochondria, and are capable of degrading multiple proteins in the type 1 interferon pathway Elliott et al.
Ns1 and Ns2 have a curious relationship with the mitochondria as optimal degradosome function appears in modified mitochondria that are shorter and more motile. The modified mitochondria possess a greater surface area and thus greater access to MAVS Goswami et al. The Ns degradosome is a heterogeneous large protein complex — kD in size. While the exact composition of the degradosome is unknown, Ns1 acts as a functional E3 ligase and to facilitate proteasomal degradation of target proteins Sun and Lopez, The RIG-I and TLR pathways both function to induce the production of interferons after contact with a PAMP pathogen-associated molecular pattern , and feed into one another's pathways due to overlapping functions.
Abstract Bovine respiratory syncytial virus RSV is a cause of respiratory disease in cattle worldwide. Publication types Review. Substances Viral Vaccines. The analysis of BRSV infection was originally hampered by its characteristic lability and poor growth in vitro. However, the advent of numerous immunological and molecular methods has facilitated the study of BRSV enormously. The knowledge gained from these studies has also provided the opportunity to develop safe, stable, attenuated virus vaccine candidates.
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