Respiratory Syncytial Virus Can Tolerate an Intergenic Sequence of at Least 160 Nucleotides with Little Effect on Transcription or Replication In Vitro and In Vivo

Ebola virus (EBOV) belongs IMPORTANCEOur current understanding of EBOV transcription regulation is limited due to the requirement for high-containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative-sense RNA viruses. A single polymerase entry site at the 3= end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from that in prototype viruses and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle.

Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Analysis of the Highly Diverse Gene Borders in Ebola Virus Reveals a Distinct Mechanism of Transcriptional Regulation

Brauburger

Boehmann

Tsuda

et al. 2014

“…However, the growth kinetics of rRSV/mGM-CSF in vitro were indistinguishable from those of rRSV/CAT, a previously described recombinant that contains a 735-nucleotide transcriptional unit encoding bacterial CAT (compared to 465 nt for the present mGM-CSF insert) inserted into the same genome locus. We have previously noted that the presence of a foreign insert attenuates RSV replication in vitro irrespective of its encoded protein (9)(10)(11)(12). The basis for this attenuation has not been characterized in detail but appears to be increased genome length.…”

Section: Construction and In Vitro Characterization Of Rrsv Expressinmentioning

confidence: 99%

Granulocyte-Macrophage Colony-Stimulating Factor Expressed by Recombinant Respiratory Syncytial Virus Attenuates Viral Replication and Increases the Level of Pulmonary Antigen-Presenting Cells

Bukreyev

Belyakov

Berzofsky

et al. 2001

Self Cite

An obstacle to developing a vaccine against human respiratory syncytial virus (RSV) is that natural infection typically does not confer solid immunity to reinfection. To investigate methods to augment the immune response, recombinant RSV (rRSV) was constructed that expresses murine granulocyte-macrophage colonystimulating factor (mGM-CSF) from a transcription cassette inserted into the G-F intergenic region. Replication of rRSV/mGM-CSF in the upper and lower respiratory tracts of BALB/c mice was reduced 23-to 74-and 5-to 588-fold, respectively, compared to that of the parental rRSV. Despite this strong attenuation of replication, the level of RSV-specific serum antibodies induced by rRSV/mGM-CSF was comparable to, or marginally higher than, that of the parental rRSV. The induction of RSV-specific CD8 ؉ cytotoxic T cells was moderately reduced during the initial infection, which might be a consequence of reduced antigen expression. Respiratory syncytial virus (RSV) is the most important viral etiologic agent of serious pediatric respiratory tract disease worldwide. RSV also is receiving increasing recognition as an important cause of respiratory tract disease in the elderly; in immunocompromised patients, such as bone marrow transplant recipients; and in the general population (reviewed in reference 16). Reinfection by RSV is common, although disease is less severe. A licensed RSV vaccine is not available, but passive immunoprophylaxis with RSV-neutralizing antibody is now available for high-risk infants (2).RSV is a nonsegmented negative-strand RNA virus of the family Paramyxoviridae. RSV encodes 11 proteins (reviewed in references 8, 15, and 16), namely, three transmembrane surface proteins (G, F, and SH); the virion matrix protein M; the nucleocapsid and polymerase proteins N, P, M2-1, and L; the putative transcription-replication regulatory factor M2-2; and two nonstructural proteins, NS1 and NS2, that have been implicated as antagonists of the type I interferon antiviral state (31). While many components of the innate and adaptive immune systems contribute to restricting and resolving an RSV infection, the increased resistance to reinfection that is conferred by prior infection appears to be mediated primarily by RSV-specific secretory and serum antibodies (16,17,27). The F and G glycoproteins are the major RSV neutralization antigens (17).In the 1960s, a formalin-inactivated RSV vaccine evaluated in infants and children was not protective and, paradoxically, was associated with increased frequency and severity of disease during subsequent natural RSV infection (26). It is now understood that the initial vaccination primed for an immunemediated pathogenic response that occurred upon reexposure to viral antigen during the subsequent natural infection, although the details of this phenomenon have not been completely elucidated. One element appears to involve the disproportionate induction of the Th2 subset of CD4 ϩ T helper lymphocytes, characterized by increased secretion of interleukin 4 (IL-4) and . Subunit...

“…This was chosen in order to facilitate the gene rearrangements, because the SH region of the complete antigenomic cDNA is unstable in bacteria and deletion of SH has only a marginal effect on virus growth. In pD53/⌬SH, the M and G genes were separated by the naturally occurring SH-G intergenic (IG) sequence (7,36). To provide a promoter-proximal insertion site, this cDNA was modified to contain a unique BlpI site in the nontranslated region of the NS1 gene immediately preceding the NS1 ORF at nt 91 to 97 relative to the 5Ј end of the encoded antigenome (Fig.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…The parental RSV strain A2 antigenomic cDNA for constructing the gene shift viruses was pD53/⌬SH, from which the SH gene had been deleted (7,36). This was chosen in order to facilitate the gene rearrangements, because the SH region of the complete antigenomic cDNA is unstable in bacteria and deletion of SH has only a marginal effect on virus growth.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

Recombinant Respiratory Syncytial Virus with the G and F Genes Shifted to the Promoter-Proximal Positions

Krempl

Murphy

Collins

2002

Self Cite

The genome of human respiratory syncytial virus (RSV) encodes 10 mRNAs and 11 proteins in the order 3-NS1-NS2-N-P-M-SH-G-F-M2-1/M2-2-L-5. The G and F glycoproteins are the major RSV neutralization and protective antigens. It seems likely that a high level of expression of G and F would be desirable for a live RSV vaccine. For mononegaviruses, the gene order is a major factor controlling the level of mRNA and protein expression due to the polar gradient of sequential transcription. In order to increase the expression of G and F, recombinant RSVs based on strain A2 were constructed in which the G or F gene was shifted from the sixth or seventh position (in a genome lacking the SH gene), respectively, to the first position (rRSV-G1/⌬SH and rRSV-F1/⌬SH, respectively). Another virus was made in which G and F were shifted together to the first and second positions, respectively (rRSV-G1F2/⌬SH). Shifting one or two genes to the promoter-proximal position resulted in increased mRNA and protein expression of the shifted genes, with G and F expression increased up to 2.4-and 7.8-fold, respectively, at the mRNA level and approximately 2.5-fold at the protein level, compared to the parental virus. Interestingly, the transcription of downstream genes was not greatly affected even though shifting G or F, or G and F together, had the consequence of moving the block of genes NS1-NS2-N-P-M-(G) one or two positions further from the promoter. The efficiency of replication of the gene shift viruses in vitro was increased up to 10-fold. However, their efficiency of replication in the lower respiratory tracts of mice was statistically indistinguishable from that of the parental virus. In the upper respiratory tract, replication was slightly reduced on some days for viruses in which G was in the first position. The magnitude of the G-specific antibody response to the gene shift viruses was similar to that to the parental virus, whereas the F-specific response was increased up to fourfold, although this was not reflected in an increase of the neutralizing activity. Thus, shifting the G and F genes to the promoter-proximal position increased virus replication in vitro, had little effect on replication in the mouse, and increased the antigen-specific immunogenicity of the virus beyond that of parental RSV.Human respiratory syncytial virus (RSV) is the leading viral cause of serious lower respiratory tract infections in infants and children worldwide. In the United States alone, RSV accounts for 73,000 to 126,000 hospitalizations of infants and children every year (15,18,32). In addition, RSV is increasingly recognized as important infectious agent in immunocompromised patients, in the elderly, and in the adult population in general. Although immunoprophylaxis by parenterally administered antibody is available for high-risk individuals, RSV lacks an effective antiviral therapy or a licensed vaccine for use in the general population.A number of live-attenuated RSV vaccine candidates have been developed by using conventional methods of biol...