Respiratory Syncytial Virus Genomic RNA Research Articles

Respiratory syncytial virus ribonucleoproteins hijack microtubule Rab11 dependent transport for intracellular trafficking.

Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.

Inhibition of RNase L and RNA-dependent Protein Kinase (PKR) by Sunitinib Impairs Antiviral Innate Immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.

Respiratory Syncytial Virus Induces Host RNA Stress Granules To Facilitate Viral Replication

Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response.

Inhibition of respiratory syncytial virus in cultured cells by nucleocapsid gene targeted deoxyribozyme (DNAzyme)

Respiratory syncytial virus (RSV), which presents the primary cause of bronchiolitis and pneumonia among infants and causes significant morbidity and mortality in immunodeficient patients, remains a health problem worldwide. Unfortunately, an effective vaccine is currently unavailable and pharmacologic treatment needs further optimization for RSV disease. Because RSV is a non-segmented negative-strand RNA virus, it may be sensitive to the genome RNA cleaving by DNAzyme, an artificial nucleic acids molecule with high catalytic capability of cleaving complementary RNA molecules. Thus, RSV-targeted DNAzymes potentially present as a therapeutic candidate of RSV diseases. In this study, DNAzymes targeting the RSV genomic RNA or mRNA were designed and synthesized, one of which (DZn1133) did cleave RSV RNA in vitro, inhibit the transcription and expression of F viral gene, reduce the RSV yield by about 7 logs and protect more than 90% RSV-infected Hep-2 cells from a cytopathic effect at 8 μM. Moreover, 10 wild RSV strains isolated from clinic patients including both subgroups A and B were all suppressed by DZn1133 with greater anti-RSV activity than antisense DNA or ribavirin.

Evidence that the Respiratory Syncytial Virus Polymerase Is Recruited to Nucleotides 1 to 11 at the 3′ End of the Nucleocapsid and Can Scan To Access Internal Signals

The 3'-terminal end of the respiratory syncytial virus genomic RNA contains a 44-nucleotide leader (Le) region adjoining the gene start signal of the first gene. Previous mapping studies demonstrated that there is a promoter located at the 3' end of Le, which can signal initiation of antigenome synthesis. The aim of this study was to investigate the role of the 3' terminus of the RNA template in (i) promoter recognition and (ii) determining the initiation site for antigenome synthesis. A panel of minigenomes containing additional sequence at the 3' end of the Le were analyzed for their ability to direct antigenome and mRNA synthesis. Minigenomes containing heterologous extensions of 6 nucleotides or more were unable to support efficient RNA synthesis. However, the activity of a minigenome with a 56-nucleotide extension could be restored by insertion of Le nucleotides 1 to 11 or 1 to 13 at the 3' end, indicating that these nucleotides, in conjunction with the 3' terminus, are sufficient to recruit polymerase to the template. Northern blot and 5' rapid amplification of cDNA ends analysis of antigenome RNA indicated that antigenome initiation occurred at the first position of Le, irrespective of the terminal extension. This finding demonstrates that the 3' terminus of the RNA is not necessary for determining the antigenome initiation site. Data are presented which suggest that following recruitment to a promoter at the 3' end of Le, the polymerase is able to scan and respond to a promoter signal embedded within the RNA template.

Potent inhibition of respiratory syncytial virus by combination treatment with 2–5A antisense and ribavirin

Respiratory syncytial virus (RSV) is a major cause of lower respiratory diseases in infants, young children, and the elderly. Ribavirin, the only currently approved drug for the treatment of RSV infections in the U.S., requires high doses to be effective. Therefore, it has only a limited clinical efficacy in the treatment of RSV infections. It has been shown that a cellular ribonuclease, RNase L, can be recruited by 2′–5′ linked tetra-adenylates (2–5A) attached to an antisense sequence complementary to the RSV genome to specifically cleave RSV genomic RNA. Here we confirm the antiviral activity of the lead 2–5A antisense compound, RBI034, by using several different viral assays. We demonstrate that RBI034 is more efficient than antisense lacking 2–5A or small interfering dsRNA (siRNA) in inhibiting RSV replication. Although the best antiviral activity of RBI034 was observed with co-treatment of RSV infection, it remained effective even when administrated 24 h after the initiation of infection. Interestingly, the activity of RBI034 can be further enhanced by a combination treatment with ribavirin. At suboptimal concentrations, neither ribavirin nor RBI034 was effective in suppressing RSV replication. However, a combination of these two drugs at the same suboptimal concentrations showed a potent inhibitory activity. The potent reduction of RSV replication by combination treatment was also confirmed in primary human airway epithelial cells. Therefore, a combination therapy of the 2–5A antisense compound RBI034 and ribavirin might be a more effective therapeutic approach for treating RSV infections than ribavirin alone.

Targeted therapy of respiratory syncytial virus in African green monkeys by intranasally administered 2-5A antisense.

Respiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants, young children, immunocompromised patients, and the institutionalized elderly. Previous work had shown that RNase L, an antiviral enzyme of the interferon system, could be recruited to cleave RSV genomic RNA by attaching tetrameric 2prime prime or minute-5prime prime or minute-linked oligoadenylates (2-5A) to an oligonucleotide complementary to repetitive gene-start sequences within the RSV genome (2-5A antisense). A 2prime prime or minute-O-methyl RNA-modified analog of the lead 2-5A anti-RSV chimera is shown here to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L- and sequence-specific manner. When administered intranasally to RSV-infected African green monkeys, this chimera reduced nasal RSV replication by up to four log(10) units in a dose- and time-dependent manner.

Rescue of a bovine respiratory syncytial virus genomic RNA analog by bovine, human and ovine respiratory syncytial viruses confirms the "functional integrity" and "cross-recognition" of BRSV cis-acting elements by HRSV and ORSV.

The nucleotide sequences of the 3' leader and 5' trailer regions were determined for genomic RNA of bovine respiratory syncytial virus (BRSV) strain A-51908. The leader and trailer sequences are '45' and '161' nucleotides in length, respectively. The functionality of BRSV leader and trailer sequences and their recognition by HRSV and ovine respiratory syncytial virus (ORSV) proteins were examined with a in vitro transcribed BRSV genomic RNA analog carrying the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of BRSV transcription signals. Upon transfection into BRSV, HRSV or ORSV infected cells, the BRSV minireplicons were 'rescued' such that the reporter gene was expressed, the minigenome was replicated and packaged into micrococcal nuclease resistant-infectious minireplicons. The passage of infectious minireplicons could be blocked by a polyclonal BRSV neutralizing antiserum. Bovine parainfluenza virus-3, a heterologous paramyxovirus was inactive in rescuing BRSV genomic RNA analog. Mutational substitution of the G residue at position 4 of leader sequence in the BRSV genomic RNA analog, with an A or U residue inhibited its transcription and replication, while replacement with a C residue had no significant effect on rescue. These results show that the cis-acting elements of BRSV are functional and are also recognized by the proteins of HRSV and ORSV. The helper virus complemented rescue system developed here will be useful for characterizing the cis-acting elements of BRSV.

Inhibition of respiratory syncytial virus replication by antisense oligodeoxyribonucleotides

Oligodeoxyribonucleotides targeted against respiratory syncytial virus (RSV) genomic RNA inhibited RSV replication in cell culture by an apparent antisense mechanism. HEp-2 cells were infected with RSV strain A2 and incubated in the presence of oligonucleotides. Virus replication was measured by enzyme-linked immunosorbent assay (ELISA), virus yield assay, or production of specific RSV mRNAs. Using ELISA, 50% effective concentration (EC 50) values were about 0.5–1 μM for an antisense oligonucleotide targeted to the start of the NS2 gene. All oligonucleotides inhibited virus antigen production as measured by ELISA. In all assays, this antisense oligonucleotide was more potent than: (1) a control oligonucleotide containing the reverse sequence; (2) oligonucleotides targeted at RSV mRNA; (3) a random sequence oligonucleotide; and (4) ribavirin. Reverse transcriptase polymerase chain reaction (RT-PCR) showed sequence-specific depletion of the genomic RNA target following treatment of cells with the antisense oligonucleotide. Specific cleavage of the genomic target RNA has been detected at the antisense oligonucleotide binding site, suggesting that cellular RNase H participates in the reaction. These results indicate that antisense oligonucleotides targeted against RSV genomic RNA can effectively inhibit RSV replication and may have therapeutic value.

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV-CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.

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.

Rescue of a 7502-Nucleotide (49.3% of Full-Length) Synthetic Analog of Respiratory Syncytial Virus Genomic RNA

A cDNA was constructed to encode an internally-truncated version of negative-sense genomic RNA (vRNA) of respiratory syncytial virus (RSV) containing (in 3′ to 5′ order) the 3′ vRNA leader region, the complement of the open reading frame for bacterial chloramphenicol acetyl transferase (CAT) under the control of RSV gene-start and gene-end signals, an intergenic nucleotide, the complete L gene including the gene-start and gene-end signals, and the 5′ vRNA trailer region. The encoded vRNA analog (RSV-CAT-L) would be 7502 nucleotides (nt) in length, 49.3% of the complete 15,222-nt parental vRNA. RSV-CAT-L vRNA was synthesized in vitro from Hga l-digested cDNA and transfected into RSV-infected cells. The vRNA was "rescued" such that it was expressed to yield CAT and was packaged into particles that could be passaged to express CAT in fresh cells. The efficiency of rescue was greatly improved by a single nucleotide substitution in the leader region that had been found to increase the efficiency of rescue and passage of the previously described 935-nt RSV-CAT vRNA. Compared to the 8-fold smaller RSV-CAT vRNA, and adjusted to molar equivalence of transfected RNA, RSV-CAT-L vRNA was 119- to 347-fold less efficient in expressing CAT upon transfection, whereas RSV-CAT-L vRNA containing the above-mentioned nucleotide substitution was 15.8-fold less efficient.

Nucleotide sequences of the 3′ leader and 5′ trailer regions of human respiratory syncytial virus genomic RNA

The nucleotide sequences of the 3′ extracistronic (leader) and 5′ extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3′ leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3′ leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5′ trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5′ trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3′ leader and 5′ trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.

Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene.

The viral genomic RNA (vRNA) of human respiratory syncytial virus is a nonsegmented negative strand that is not infectious alone. To develop methods for complementing synthetic vRNA with viral proteins, a cDNA was constructed to encode a vRNA in which all of the viral protein-coding sequences were removed and replaced with a negative-sense copy of the bacterial chloramphenicol acetyltransferase gene. Upon transfection into respiratory syncytial virus-infected cells, the synthetic vRNA was "rescued" such that it was amplified, expressed, and packaged into infectious virions. A heterologous paramyxovirus, parainfluenza virus 3, was inactive in rescue. Further internal deletions mapped the cis-acting viral sequences required for rescue to two segments totaling 105 nucleotides (nt) derived from the two vRNA ends. Rescue was unaffected by replacement of the 44-nt 3'-terminal leader region with a 50-nt sequence that is complementary to the 5' terminus and represents the 3' end of the positive-sense replicative intermediate RNA. This 5'-end complement was related to the parental leader region only near the 3' terminus (91% or 73% identical for the first 11 or 22 nt, respectively). The addition of 11 heterologous nt to the 3' end of the parental leader region ablated rescue, suggesting that the 3'-proximal conserved domain is required and cannot function from an internal site. However, deletion of the 3'-terminal 3 nt, or a double transition at positions 4 and 5, had no effect on rescue. Thus, the 3'-terminal 5 nt, although conserved between 3' ends of the negative- and positive-sense RNAs, do not appear to be essential.

The 1B (NS2), 1C (NS1) and N Proteins of Human Respiratory Syncytial Virus (RSV) of Antigenic Subgroups A and B: Sequence Conservation and Divergence within RSV Genomic RNA

A 2330 nucleotide sequence spanning the 1B (NS2), IC (NS1) and N genes and intergenic regions of human respiratory syncytial virus strain 18537, representing antigenic subgroup B, was determined by sequencing cloned cDNAs of intracellular mRNAs. Comparison with the previously reported sequences for strain A2 of subgroup A showed that 1B, 1C and N were highly conserved at the nucleotide level (78, 78 and 86% identity, respectively) and at the amino acid level (92, 87 and 96% identity, respectively). The gene-start signals were exactly conserved between subgroups, and the gene-end signals contained only a single nucleotide substitution each in 1B and N. In most cases intergenic and non-coding gene sequences that were not part of presumed transcriptive signals were much less well conserved (generally 50 to 71%) than sequences that were part of translational open reading frames (82 to 86%). The nucleotide and deduced amino acid sequences of the N gene and protein of the Long strain of subgroup A were determined by sequencing cDNA clones of intracellular mRNA; the nucleotide sequence (representing all but the first 10 nucleotides of the gene) contained 15 differences from that of the A2 strain, but the deduced amino acid sequences were identical.