Paramyxovirus persistence

In this study, SV5 infection of Balb/c mouse fibroblast cells has been used as a model system to investigate the possible mechanisms underlying the establishment and maintenance of Paramyxovirus persistence. It was found that following entry to these cells, the virus initiated a wave of transcription and replication, similar to that of a permissive infection, in which normal levels of each of the virus proteins were synthesized. However, by 48-72 hours post infection (p.i.) there was an almost complete cessation of virus mRNA and protein synthesis. Despite the decrease in virus activity, full length viral genome RNA and P and NP, the proteins involved in transcription and replication, could be detected at consistently high levels up to 5 days p.i., although the levels of HN, M, F and V declined. Immunofluorescence analysis supported these data showing that at later times p.i. although there were some cells positive for all the viral proteins, a high proportion of cells were strongly positive for NP, L and P, but negative for M, F and HN. In these cells, NP, L and P were often located in discrete cytoplasmic foci. These results suggested that the persistently infected cell population consisted of some cells in which the virus was active and other in which it was quiescent within cytoplasmic inclusions. A series of cell lines was established from a monolayer of Balb/c cells that had been infected at a high multiplicity. Immunofluorescence studies showed only a minority of cells in these clones to be infected with virus, indicating that during division, not all daughter cells became infected. Of the infected cells, some were positive for all the viral proteins, while others were positive for only NP and P. Co-cultivation of the cloned cells with Vero cells, which are permissive for SV5 replication, rapidly yielded non-defective virus, suggesting that the virus was active in some cells. These results suggested that the persisting virus was in a state of flux, able to reside as inclusions of inactive nucleocapids from which it could reactivate to initiate a new round of infection. Experiments aiming to determine if the persistently infected cells were resistant to immune attack demonstrated that cells at 5 days p.i., in which the majority of cells were quiescently infected, were less susceptible to immune lysis than cells at 1 day p.i. in which there was ongoing protein synthesis. Further experiments were carried out both to try to determine what had induced the persistent state in mouse cells and also to examine factors which might induce a similar state in different cell lines

Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines

Investigating the Influence of Ribavirin on Human Respiratory Syncytial Virus RNA Synthesis by Using a High-Resolution Transcriptome Sequencing Approach

Human respiratory syncytial virus (HRSV) is a major cause of serious respiratory tract infection. Treatment options include administration of ribavirin, a purine analog, although the mechanism of its anti-HRSV activity is unknown. We used transcriptome sequencing (RNA-seq) to investigate the genome mutation frequency and viral mRNA accumulation in HRSV-infected cells that were left untreated or treated with ribavirin. In the absence of ribavirin, HRSV-specific transcripts accounted for up to one-third of total RNA reads from the infected-cell RNA population. Ribavirin treatment resulted in a>90% reduction in abundance of viral mRNA reads, while at the same time no such reduction was detected for the abundance of cellular transcripts. The presented data reveal that ribavirin significantly increases the frequency of HRSV-specific RNA mutations, suggesting a direct influence on the fidelity of the HRSV polymerase. The presented data show that transitions and transversions occur during HRSV replication and that these changes occur in hot spots along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, our data indicate that in the continuous cell types used and at the time points analyzed, the abundances of some HRSV mRNAs do not reflect the order in which the mRNAs are transcribed

Helical ordering of envelope‐associated proteins and glycoproteins in respiratory syncytial virus

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly

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