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In a prototypical response to an acute viral infection it would be expected that the adaptive immune response would eliminate all virally infected cells within a few weeks of infection. However many (non-retrovirus) RNA viruses can establish 'within host' persistent infections that occasionally lead to chronic or reactivated disease. Despite the importance of 'within host' persistent RNA virus infections, much has still to be learnt about the molecular mechanisms by which RNA viruses establish persistent infections, why innate and adaptive immune responses fail to rapidly clear these infections, and the epidemiological and potential disease consequences of such infections.

It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-β promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-β promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system.

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

A panel of influenza A viruses encoding mutant NS1 proteins was created in which a number of NS1 functions, including interactions with dsRNA, PI3K, CPSF30 and PKR, were inhibited. Surprisingly, given previous reports that NS1 activates PI3K to prevent apoptosis, the mutant viruses rUd-Y89F and rUd-P164/7A that fail to activate PI3K did not induce any more apoptosis than wild-type virus in MRC-5 and A549 cells, even though these cells are highly sensitive to inducers of apoptosis. Induction of cell death by the apoptogenic rUd-184-8(P) virus could not be prevented by serum-mediated activation of PI3K/Akt. Neither infection of MRC-5 or A549 cells with wild-type virus nor constitutive expression of NS1 prevented cell death caused by apoptosis inducers, suggesting that NS1 is not directly anti-apoptotic. Our data suggest that the loss of a functionally intact NS1 protein promotes apoptosis, but this is not due to an inability to activate PI3K.

Posttranslational modification of viral proteins by cellular enzymes is a feature of many virus replication strategies. Here, we report that during infection the multifunctional human influenza A virus NS1 protein is phosphorylated at threonine-215. Substitution of alanine for threonine at this position reduced early viral propagation, an effect apparently unrelated to NS1 antagonizing host interferon responses or activating phosphoinositide 3-kinase signaling. In vitro, a subset of cellular proline-directed kinases, including cyclin dependent kinases (CDKs) and extracellular signal-regulated kinases (ERKs), potently phosphorylated NS1 protein at threonine-215. Our data suggest that CDK/ERK-mediated phosphorylation of NS1 at threonine-215 is important for efficient virus replication.

Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85beta regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85beta. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85beta is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85beta and NS1. Equivalent residues in p85alpha have previously been implicated in the basal inhibition of p110. However, such p85alpha residues were unable to substitute for those in p85beta with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85beta inter-SH2 domain.

We have previously reported that the addition of interferon (IFN) to the culture medium of Vero cells (which cannot produce IFN) that were infected with the CPI− strain of parainfluenza virus 5 (PIV5, formally known as SV5), that fails to block IFN signaling, rapidly induces alterations in the relative levels of virus mRNA and protein synthesis. In addition, IFN treatment also caused a rapid redistribution of virus proteins and enhanced the formation of cytoplasmic viral inclusion bodies. The most studied IFN-induced genes with known anti-viral activity are MxA, PKR and the Oligo A synthetase/RNase L system. We therefore examined the effects of these proteins on the replication cycle of PIV5. These studies revealed that while these proteins had some anti-viral activity against PIV5 they were not primarily responsible for the very rapid alteration in virus protein synthesis observed following IFN treatment, nor for the IFN-induced formation of virus inclusion bodies, in CPI− infected cells.

The induction of IFN-beta by the paramyxovirus PIV5 (formerly known as SV5) is limited by the action of the viral V protein that targets the cellular RNA helicase mda-5. Here we show that 12 other paramyxoviruses also target mda-5 by a direct interaction between the conserved cysteine-rich C-terminus of their V proteins and the helicase domain of mda-5. The inhibition of IFN-beta induction is not species-restricted, being observed in a range of mammalian cells as well as in avian cells, and we show that the inhibition of mda-5 function is also not restricted to mammalian cells. In contrast, the V proteins do not bind to the related RNA helicase RIG-I and do not inhibit its activity. The relative contributions of mda-5 and RIG-I to IFN-beta induction are discussed.

In this article we show that the paramyxovirus SV5 is a poor inducer of interferon-beta (IFN-beta). This inefficient induction is a consequence of the expression of an intact viral V protein. In the absence of the viral V protein cysteine-rich C-terminal domain, IFN-beta mRNA is strongly induced and the transcription factors NF-kappaB and IRF-3 are activated significantly. The V protein can work in isolation from SV5 to block intracellular dsRNA signaling. The mechanism of block to dsRNA signaling is distinct from that previously observed for blocking IFN signaling in that proteolysis of candidate factors cannot be detected, and furthermore, the respective blocks require distinct protein domains. Blocking of the induction of IFN-beta by dsRNA requires the C-terminal cysteine-rich domain, a feature that is highly conserved among paramyxoviruses. We demonstrate that the V proteins from other paramyxoviruses have equivalent functions and speculate that limiting the yield of IFN-beta during infection may be a general property of paramyxoviruses.

CPI(+) and CPI(-) are two canine isolates of simian virus 5 (SV5). CPI(+) was originally isolated from the cerebrospinal fluid of a dog with temporary posterior paralysis and CPI(-) was recovered at 12 days p.i. from the brain tissue of a dog experimentally infected with CPI(+). We have previously shown that the V protein of SV5 blocks interferon (IFN) signalling by targeting STAT1 for degradation. Here we report that whilst CPI(+) targets STAT1 for degradation, CPI(-) fails to and as a consequence, CPI(+) blocks IFN signalling but CPI(-) does not. Three amino acid differences in the P/V N-terminal common domain of the V protein are responsible for the observed difference in the abilities of CPI(+) and CPI(-) to block IFN signalling. In cells persistently infected with CPI(-) the virus may become repressed in response to IFN, under which circumstances virus glycoproteins are lost from the surface of infected cells and virus nucleocapsid proteins accumulate in cytoplasmic inclusion bodies. We suggest that in vivo cells infected with IFN-resistant viruses (in which there would be continuous virus protein synthesis) may be more susceptible to killing by cytotoxic T cells than cells infected with IFN-sensitive viruses (in which virus protein synthesis was repressed), and a model of virus persistence is put forward in which there is alternating selection of IFN-resistant and IFN-sensitive viruses depending upon the state of the adaptive immune response.

STAT1 and STAT2 are cellular transcription factors involved in interferon (IFN) signaling and are thus critical for the IFN-induced antiviral state. We have previously shown that the paramyxovirus Simian Virus 5 (SV5) blocks both type I and type II interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. To determine whether this is a feature common to all Paramyxoviridae, we examined the abilities of SV5, Sendai virus (SeV), human respiratory syncytial virus (RSV), and human parainfluenza viruses types 2 and 3 (hPIV2 and hPIV3, respectively) to block interferon signaling. The results showed that in reporter assays SV5, SeV, and hPIV3 blocked both type I and type II IFN-signaling; hPIV2 blocked type I but not type II IFN-signaling; and RSV failed to block either type I or type II IFN-signaling. In agreement with these results, SV5 and SeV inhibited the formation of the ISGF3 and GAF transcription complexes (essential for type I and type II signaling, respectively). Surprisingly, although hPIV3 inhibited IFN-induction of the ISGF3 complex, GAF complexes were detected in hPIV3-infected cells. hPIV2 also blocked the formation of the ISGF3 complex but not the GAF complex, whereas RSV failed to block the induction of either complex. SV5 was the only virus that caused the degradation of STAT1. Indeed, in SeV- and hPIV3-infected cells STAT1 was phosphorylated on tyrosine 701 (Y701), a characteristic of IFN receptor activation. However, consistent with these viruses blocking IFN signaling downstream of receptor activation, there was a specific reduction in the levels of serine 727 (S727)-phosphorylated forms of STAT1α in SeV- and hPIV3-infected cells. In contrast both (Y701)- and (S727)-phosphorylated forms of STAT1 were detected in hPIV2-infected cells but there was a specific loss of STAT2. Both STAT1 (including Y701- and S727-phosphorylated forms) and STAT2 could readily be detected in RSV-infected cells. Despite not being able to block type I or type II IFN signaling, RSV was able to replicate in human cells that produce and respond to IFN, suggesting that RSV must have an alternative method(s) for circumventing the IFN response. These results demonstrate that, although interference with IFN signaling is a common strategy among Paramyxovirinae, distinct virus-specific mechanisms are used to achieve this end.

The monoclonal antibody (mAb) SV5-Pk is used widely in a variety of procedures to detect recombinant proteins tagged with the Pk tag, a 14 amino acid sequence derived from the P and V proteins of the paramyxovirus Simian Virus 5. Here we report on the isolation and characterisation of four additional SV5-Pk mAbs (termed SV5-Pk2 to 5) that bind the Pk tag. All the SV5-Pk mAbs can detect Pk tagged recombinant proteins in a variety of immunological procedures, including ELISA and immunofluorescence. Using SPOT technology, the minimal binding epitope of each SV5-Pk mAb was defined by one-sided terminal truncation analysis from either the amino- or carboxy-ends of the Pk peptide. Each mAb recognises slightly different epitopes within the Pk tag, ranging from 5 to 9 amino acids in length. The equilibrium dissociation constants (Kd) of the mAbs, as measured by surface plasmon resonance, ranged from approximately 20 to 60 pmol. Cysteine scanner mutations throughout the Pk tag revealed that some amino acids within the minimal binding epitopes were critical for mAb binding, while others could readily be substituted with little or no effect on antibody binding. The development of the Pk tag as a spacer arm for site-directed chemical coupling, and the use of the mAbs to monitor purification and coupling procedures, is discussed.

To facilitate site-directed chemical coupling of antigens to the heat labile enterotoxin B subunit of Escherichia coli (LTB), a series of genetically modified fusion proteins of LTB were constructed. The LTB fusion proteins had modified versions of a short (14 amino acid) spacer epitope called the Pk tag attached at their C termini. The LTB-Pk.cys mutants differed from each other in the position of a single cysteine residue within the Pk tag. The presence of a cysteine residue at any position within the Pk spacer tag did not prevent the LTB-Pk.cys proteins from forming pentamers or binding to GM1 gangliosides, but the position of the cysteine had variable impact on the yield of the fusion proteins. Following site-directed chemical coupling of antigens to the cysteine residue within the Pk tag, the LTB–antigen conjugates retained their ability to bind GM1 on the surface of eukaryotic cells. Intranasal immunisation of mice with an experimental antigen (HRP) chemically linked to LTB-Pk.cys induced high levels of anti-HRP antibodies that could be detected in the serum, saliva and nasal and lung washes. No antibody responses to HRP were detected when HRP was co-administered with, but not linked to, LTB-Pk.cys.

A series of vectors is described which enables the episomal expression of proteins fused to different tag sequences in Schizosaccharomyces pombe. Proteins can be expressed with their amino termini fused to GFP/EGFP, three copies of the HA or Pk epitopes or a combined tag which contains two copies of the myc epitope and six histidine residues (MH). Fusion of the carboxyl terminus of a protein to a tag is possible with GFP/EGFP or Pk. Expression of the fusion proteins is controlled by the medium strength mutant version of the regulatable nmt1 promoter.

Using recombinant proteins extracted from mammalian cells, in a novel protein:protein binding assay, direct interaction of the nucleoprotein (NP) of simian virus 5 with the phosphoprotein (P) and V protein (V) was demonstrated. The amount of NP bound by V was found to be significantly less than that bound by P. Furthermore, preabsorption of NP with P removed the fraction of NP that could be bound by V, but preabsorption of NP with V did not remove all the NP that could be bound by P. These results suggested that V bound a subpopulation of the NP recognised by P. Further analysis revealed that P bound both soluble and homopolymeric forms of NP, while V bound only the soluble form; thus demonstrating that the binding sites on P and V, for soluble NP, are located within the N-terminal domain common to both P and V proteins. A monoclonal antibody, which recognised an epitope in the unique C-terminus of P, blocked the binding of P to polymeric NP but not to soluble NP. These results also suggest that there are two binding sites on NP for P; the site that interacts with the P/V common domain being either hidden or conformationally altered in polymeric NP.

Monoclonal antibodies were generated against the feline homologue of CD4 (fCD4) by immunisation of mice with solid matrix antigen-antibody complexes of monoclonal antibody Fel7 (anti-fCD4) and formalin-fixed Staphylococcus A (SMAA-fCD4). The resulting fusion produced nine monoclonal antibodies each of which recognised a major population of feline lymphocytes and which immunoprecipitated a 55 kDa ligand from the feline T lymphosarcoma cell line 3201. Epitope mapping of the antibodies against soluble fCD4 by surface plasmon resonance indicated that the antibodies recognised five separate epitopes distinct from that defined by the Fel7 antibody used to prepare the SMAA-fCD4. These data demonstrate that SMAA complexes are an efficient means of generating monoclonal antibodies recognising novel epitopes on an antigen. One monoclonal antibody (vpg39) recognised an epitope that was expressed variably between cats, being either present or completely absent. Analysis of peripheral blood lymphocytes from specific pathogen free cats suggested that failure to react with the vpg39 antibody was an inherited trait.

For successful vaccination to many diseases, it is necessary to induce both humoral and cell-mediated immune responses to the infectious agent: this may require the incorporation of multiple antigens from the same microbe into the vaccine. In this article, Richard Randall proposes that one of the most practical and effective ways of producing multivalent vaccines may be through the construction of solid matrix-antibody-antigen (SMAA) complexes. The advantages of such vaccines and their future potential is discussed.

A method is described for forming monolayers of Staphylococcus aureus Cowan strain A on plastic tissue culture plates (A plates). Bacteria remain bound to such plates even under strong protein denaturing conditions. The specific binding of antibody to A plates provides a rapid method for immune precipitation on a solid matrix. Antibody can be covalently crosslinked to staphylococcus monolayers by fixation with paraformaldehyde without significant loss of specific antigen binding capacity. Furthermore antigen-antibody complexes may be efficiently disrupted with 9 M urea and non-ionic detergent and both antibody and antigen renatured by removal of urea, antibody regaining the ability to bind fresh antigen. In the presence of 9 M urea and 1% Nonidet P-40 fixed antibody remains bound to plates while antigen is released into the urea solution, providing a method for the immunological purification of proteins. Plates with such fixed antibody may be used multiple times to bind antigen. The use of this method is illustrated by the purification of 2 adenovirus structural proteins and a herpes simplex virus glycoprotein, by means of specific monoclonal antibodies to these proteins crosslinked to A plates. A method is also desribed to enrich for functional antibody from immune precipitates by dissociating antibody-antigen complexes with 9 M urea and 1% Nonidet P-40 and isolating the antigen free antibody on Staphylococcus aureus Cowan strain A. A plates have also been used to separate suspension cells on the basis of their cell surface antigens (e.g., lymphocyte subpopulations or cells expressing virus antigens on their surface). Cells were either (1) reacted with specific antisera to cell surface determinants and panned over A plates, cells with antibody on their surface binding to the monolayer, or (2) panned over A plates to which specific antibody had been coupled. Separated cells may be probed directly for certain properties, such as their ability to support virus replication. The staphylococcus monolayer does not prevent the growth of tissue culture cells on the plastic surface and the selected cell population can be examined for cells giving rise to infectious centres by the subsequent addition of permissive cells.