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Cytoplasmic inclusion bodies (IBs) are a common feature of single-stranded, non-segmented, negative-strand RNA virus (Mononegavirales) infections and are thought to be regions of active virus transcription and replication. Here we followed the dynamics of IB formation and maintenance in cells infected with persistent and lytic/acute variants of the paramyxovirus, parainfluenza virus type 5 (PIV5). We show that there is a rapid increase in the number of small inclusions bodies up until approximately 12 h post-infection. Thereafter the number of inclusion bodies decreases but they increase in size, presumably due to the fusion of these liquid organelles that can be disrupted by osmotically shocking cells. No obvious differences were observed at these times between inclusion body formation in cells infected with lytic/acute and persistent viruses. IBs are also readily detected in cells persistently infected with PIV5, including in cells in which there is little or no ongoing virus transcription or replication. In situ hybridization shows that genomic RNA is primarily located in IBs, whilst viral mRNA is more diffusely distributed throughout the cytoplasm. Some, but not all, IBs show incorporation of 5-ethynyl-uridine (5EU), which is integrated into newly synthesized RNA, at early times post-infection. These results strongly suggest that, although genomic RNA is present in all IBs, IBs are not continuously active sites of virus transcription and replication. Disruption of IBs by osmotically shocking persistently infected cells does not increase virus protein synthesis, suggesting that in persistently infected cells most of the virus genomes are in a repressed state. The role of IBs in PIV5 replication and the establishment and maintenance of persistence is discussed.

Parainfluenza virus type 5 (PIV5) can either have a persistent or a lytic phenotype in cultured cells. We have previously shown that the phenotype is determined by the phosphorylation status of the phosphoprotein (P). Single amino acid substitutions at critical residues, including a serine-to-phenylalanine substitution at position 157 on P, result in a switch between persistent and lytic phenotypes. Here, using PIV5 vectors expressing either mCherry or GFP with persistent or lytic phenotypes, we show that in co-infections the persistent phenotype is dominant. Thus, in contrast to the cell death observed with cells infected solely with the lytic variant, in co-infected cells persistence is immediately established and both lytic and persistent genotypes persist. Furthermore, 10-20 % of virus released from dually infected cells contains both genotypes, indicating that PIV5 particles can package more than one genome. Co-infected cells continue to maintain both genotypes/phenotypes during cell passage, as do individual colonies of cells derived from a culture of persistently infected cells. A refinement of our model on how the dynamics of virus selection may occur in vivo is presented.

NS1 proteins of influenza A and B viruses share limited sequence homology, yet both are potent manipulators of host cell processes, particularly interferon (IFN) induction. Although many cellular partners are reported for A/NS1, only a few (e.g. PKR and ISG15) have been identified for B/NS1. Here, affinity-purification and mass spectrometry were used to expand the known host interactome of B/NS1. We identified 22 human proteins as new putative targets for B/NS1, validating several, including DHX9, ILF3, YBX1 and HNRNPC. Consistent with two RNA-binding domains in B/NS1, many of the identified factors bind RNA and some interact with B/NS1 in an RNA-dependent manner. Functional characterization of several B/NS1 interactors identified SNRNP200 as a potential positive regulator of host IFN responses, while ILF3 exhibited dual roles in both IFN induction and influenza B virus replication. These data provide a resource for future investigations into the mechanisms underpinning host cell modulation by influenza B virus NS1.

Deletion or truncation of NS1, the principal IFN antagonist of influenza viruses, leads to increased IFN induction during influenza virus infection. We have studied activation of the IFN induction cascade by both wild-type and NS1-defective viruses at the single-cell level using a cell line expressing GFP under the control of the IFN-β promoter and by examining MxA expression. The IFN-β promoter was not activated in all infected cells even during NS1-defective virus infections. Loss of NS1 expression is therefore insufficient per se to induce IFN in an infected cell, and factors besides NS1 expression status must dictate whether the IFN response is activated. The IFN response was efficiently stimulated in these cells following infection with other viruses; the differential IFN response we observe with influenza viruses is therefore not cell specific but is likely due to differences in the nature of the infecting virus particles and their subsequent replication.

Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2'-O-methyltransferase activity, an enzyme that methylates the 2'-hydroxyl group of ribose sugars in the 5'-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2'-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5.

Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-β promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade.

Although the Enders strain of mumps virus (MuV) encodes a functional V protein that acts as an interferon (IFN) antagonist, in multi-cycle growth assays MuV Enders grew poorly in naïve ('IFN-competent' Hep2) cells but grew to high titres in 'IFN-compromised' Hep2 cells. Even so, the growth rate of MuV Enders was significantly slower in 'IFN-compromised' Hep2 cells when compared with its replication rate in Vero cells and with the replication rate of parainfluenza virus type 5 (a closely related paramyxovirus) in both naïve and 'IFN-compromised' Hep2 cells. This suggests that a consequence of slower growth is that the IFN system of naïve Hep2 cells can respond quickly enough to control the growth of MuV Enders. This is supported by the finding that rapidly growing variants of MuV Enders that were selected on 'IFN-compromised' Hep2 cells (i.e. in the absence of any selection pressure exerted by the IFN response) also grew to high titres on naïve Hep2 cells. Sequencing of the complete genome of one of these variants identified a single point mutation that resulted in a substitution of a conserved asparagine by histidine at position 498 of the haemagglutinin-neuraminidase protein, although this mutation was not present in all rapidly growing variants. These results support the concept that there is a race between the ability of a cell to detect and respond to virus infection and the ability of a virus to block the IFN response. Importantly, this emphasizes that factors other than viral IFN antagonists influence the sensitivity of viruses to IFN.

Although the replication cycle of parainfluenza virus type 5 (PIV5) is initially severely impaired in cells in an interferon (IFN)-induced antiviral state, the virus still targets STAT1 for degradation. As a consequence, the cells can no longer respond to IFN and after 24-48 h, they go out of the antiviral state and normal virus replication is established. Following infection of cells in an IFN-induced antiviral state, viral nucleocapsid proteins are initially localized within small cytoplasmic bodies, and appearance of these cytoplasmic bodies correlates with the loss of STAT1 from infected cells. In situ hybridization, using probes specific for the NP and L genes, demonstrated the presence of virus genomes within these cytoplasmic bodies. These viral cytoplasmic bodies do not co-localize with cellular markers for stress granules, cytoplasmic P-bodies or autophagosomes. Furthermore, they are not large insoluble aggregates of viral proteins and/or nucleocapsids, as they can simply and easily be dispersed by 'cold-shocking' live cells, a process that disrupts the cytoskeleton. Given that during in vivo infections, PIV5 will inevitably infect cells in an IFN-induced antiviral state, we suggest that these cytoplasmic bodies are areas in which PIV5 genomes reside whilst the virus dismantles the antiviral state of the cells. Consequently, viral cytoplasmic bodies may play an important part in the strategy that PIV5 uses to circumvent the IFN system.

The non-structural (NS1) protein of influenza A viruses is a non-essential virulence factor that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced proteins, such as dsRNA-dependent protein kinase R (PKR) and 2'5'-oligoadenylate synthetase (OAS)/RNase L. However, it is clear that NS1 also acts directly to modulate other important aspects of the virus replication cycle, including viral RNA replication, viral protein synthesis, and general host-cell physiology. Here, we review the current literature on this remarkably multifunctional viral protein. In the first part of this article, we summarize the basic biochemistry of NS1, in particular its synthesis, structure, and intracellular localization. We then discuss the various roles NS1 has in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. We focus on the NS1-RNA and NS1-protein interactions that are fundamental to these processes, and highlight apparent strain-specific ways in which different NS1 proteins may act. In this regard, the contributions of certain NS1 functions to the pathogenicity of human and animal influenza A viruses are also discussed. Finally, we outline practical applications that future studies on NS1 may lead to, including the rational design and manufacture of influenza vaccines, the development of novel antiviral drugs, and the use of oncolytic influenza A viruses as potential anti-cancer agents.

The interferon (IFN) system is an extremely powerful antiviral response that is capable of controlling most, if not all, virus infections in the absence of adaptive immunity. However, viruses can still replicate and cause disease in vivo, because they have some strategy for at least partially circumventing the IFN response. We reviewed this topic in 2000 [Goodbourn, S., Didcock, L. & Randall, R. E. (2000). J Gen Virol 81, 2341-2364] but, since then, a great deal has been discovered about the molecular mechanisms of the IFN response and how different viruses circumvent it. This information is of fundamental interest, but may also have practical application in the design and manufacture of attenuated virus vaccines and the development of novel antiviral drugs. In the first part of this review, we describe how viruses activate the IFN system, how IFNs induce transcription of their target genes and the mechanism of action of IFN-induced proteins with antiviral action. In the second part, we describe how viruses circumvent the IFN response. Here, we reflect upon possible consequences for both the virus and host of the different strategies that viruses have evolved and discuss whether certain viruses have exploited the IFN response to modulate their life cycle (e.g. to establish and maintain persistent/latent infections), whether perturbation of the IFN response by persistent infections can lead to chronic disease, and the importance of the IFN system as a species barrier to virus infections. Lastly, we briefly describe applied aspects that arise from an increase in our knowledge in this area, including vaccine design and manufacture, the development of novel antiviral drugs and the use of IFN-sensitive oncolytic viruses in the treatment of cancer.

Mapuera virus (MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genus Rubulavirus on the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.

Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture, to aid in growth of the virus, a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5, which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.

Previous work has documented that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation, whilst the V protein of human parainfluenza virus type 2 (hPIV2) targets STAT2. Here, it was shown that the processes of ubiquitination and degradation could be reconstructed in vitro by using programmed rabbit reticulocyte lysates. Using this system, the addition of bacterially expressed and purified SV5 V protein to programmed lysates was demonstrated to result in the polyubiquitination and degradation of in vitro-translated STAT1, but only if human STAT2 was also present. Surprisingly, in the same assay, purified hPIV2 V protein induced the polyubiquitination of both STAT1 and STAT2. In the light of these in vitro results, the specificity of degradation of STAT1 and STAT2 by SV5 and hPIV2 in tissue-culture cells was re-examined. As previously reported, STAT1 could not be detected in human cells that expressed SV5 V protein constitutively, whilst STAT2 could not be detected in human cells that expressed hPIV2 V protein, although the levels of STAT1 may also have been reduced in some human cells infected with hPIV2. In contrast, STAT1 could not be detected, whereas STAT2 remained present, in a variety of animal cells, including canine (MDCK) cells, that expressed the V protein of either SV5 or hPIV2. Thus, the V protein of SV5 appears to be highly specific for STAT1 degradation, but the V protein of hPIV2 is more promiscuous.

Sequence comparison of the V/P and F genes of 13 human, canine, porcine and simian isolates of simian virus 5 (SV5) revealed a surprising lack of sequence variation at both the nucleotide and amino acid levels (0-3%), even though the viruses were isolated over 30 years and originated from countries around the world. Furthermore, there were no clear distinguishing amino acid or nucleotide differences among the isolates that correlated completely with the species from which they were isolated. In addition, there was no evidence that the ability of the viruses to block interferon signalling by targeting STAT1 for degradation was confined to the species from which they were isolated. All isolates had an extended cytoplasmic tail in the F protein, compared with the original W3A and WR monkey isolates. Sequence analysis of viruses that were derived from human bone-marrow cells isolated in London in the 1980s revealed that, whilst they were related more closely to one another than to the other isolates, they all had identifying differences, suggesting that they were independent isolates. These results therefore support previous data suggesting that SV5 can infect humans persistently, although the relationship of SV5 to any human disease remains highly contentious. Given that SV5 has been isolated on multiple occasions from different species, it is proposed that the term simian virus 5 is inappropriate and suggested that the virus should be renamed parainfluenza virus 5.

Following infection of BALB/c fibroblastic (BF) cells with simian virus 5 (SV5) only low levels of infectious virus were produced and the majority of cells survived the infection. However at 1 day post-infection (p.i.), near normal levels of all the virus proteins were synthesized and the virus genome was replicated. RNA analysis of the infected cells revealed that the levels of viral genomic RNA remained high over 5 days of infection, but that viral mRNA levels were significantly reduced by 3 days p.i. There was no evidence for the accumulation of defective genomes over this period. The reduction in mRNA levels was reflected by a concomitant decrease in the rate of ongoing viral protein synthesis. Despite the apparent decrease in viral transcription, comparative measurements of the relative levels of the different virus proteins at various times p.i. revealed that the levels of the P and NP proteins were similar at 1 and 5 days p.i. but the levels of V, M and F declined. Immunofluorescence analysis supported this data showing that at later times p.i., although there were some cells which were positive for all the viral proteins, a high proportion of cells were strongly positive for NP and P but negative for M, F and HN proteins. In these cells, NP and P were often located in discrete cytoplasmic foci. A series of cell lines were established from BF cells that had been infected at high multiplicity. Immunofluorescence studies showed that only a minority of cells in these cell lines were infected. This suggests that upon cell division, in a proportion of cells, virus replication was not taking place; otherwise it would be expected that all the daughter cells would remain infected. However, upon co-cultivation of these cells with Vero cells (cells that are fully permissive for SV5 replication), non-defective virus could be recovered. Virus cytoplasmic inclusion bodies could still be detected in a small proportion of BF cells that had been infected at high m.o.i. and passaged 10 times over a 12 week period, and again low levels of infectious virus could be recovered from these cells. It is proposed that in these persistently infected cells, the majority of virus genomes reside in an inactive form in cytoplasmic inclusion bodies but from which virus may occasionally be reactivated.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously shown that immunization with solid matrix-antigen-antibody (SMAA) complexes induces both vigorous humoral and cell-mediated immune responses and have suggested that this method of vaccination may be developed for use in humans, and potentially as a vaccine against AIDS. Here we demonstrate that a small oligopeptide can act as a tag for the construction of SMAA complexes using a tag-specific monoclonal antibody and tag-linked antigens. We show that a 14-amino acid oligopeptide, present in the phospho (P) and V proteins of simian virus 5 (SV5), retains its antigenicity when attached to the C terminus of three 'foreign' proteins [p27 and gp110 of simian immunodeficiency virus (SIV) and glutathione S-transferase] such that these proteins can be incorporated into SMAA complexes using a monoclonal antibody (MAb) that was originally raised against the native SV5 P and V proteins. Mice were immunized with SMAA complexes containing recombinant p27-TAG and MAbs have been isolated that recognized native SIV p27. The significance of these results in terms of the development of SMAA complexes as human vaccines is discussed.

The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene was determined for a simian (W3), human (LN) and two canine (CPI+/CPI-) isolates of simian virus 5 (SV5). A comparison of the predicted amino acid sequences revealed that the human and canine isolates varied from the simian isolate by 1.7% and 2.4% respectively. This lack of significant variation between the HN proteins of the four SV5 isolates suggests that insufficient differences have occurred between isolates to confine them to a specific host.

Two canine isolates of simian virus 5 (SV5), termed CPI+ and CPI-, were examined for their ability to react with a bank of monoclonal antibodies (MAbs) that had been previously raised against a human isolate of SV5. CPI- virus was originally isolated from the brain of a gnotobiotic dog infected with CPI+ virus and establishes persistent infections more readily than CPI+ in vitro. Of more than 50 MAbs tested, only one (P-k) reacted with CPI+ but not CPI-, enabling distinction between the two canine isolates. It had been shown previously that MAb P-k reacts with an epitope common to both the P and V proteins. In order to characterize further the epitope binding site of this MAb the P/V genes of CPI+ and CPI- were sequenced. There were four nucleotide differences between CPI+ and CPI-, three of which resulted in predicted amino acid substitutions. Synthetic peptides corresponding to regions encompassing these changes were made and radioimmune competition assays were used to identify the epitope binding site of MAb P-k. Sequence comparison of the P/V gene of CPI+ with the published sequence of a monkey isolate of SV5 (W3) revealed 14 nucleotide differences with five amino acid substitutions. The only amino acid substitution observed between CPI+, CPI- and W3 which altered the predicted secondary structures of the P and V proteins was a leucine to proline change that induced a predicted beta-turn and resulted in the loss of binding of MAb P-k.

Transcripts from two immediate early (IE) genes have been identified in cells infected with the gamma herpesvirus, herpesvirus saimiri. One is a 1.3 kb RNA transcribed from the HindIII-G fragment of virus DNA (IE-G), the other is a 1.6 kb RNA from the gene for the IE 52K phosphoprotein. Labelled oligonucleotide probes specific for each of these RNAs have been used in in situ hybridization experiments to compare their expression in individual cells in infected populations. In the presence of cycloheximide, the IE-G RNA accumulates synchronously throughout the population of infected cells and prior to the asynchronous accumulation of RNA from the gene for the IE 52K protein in the same population of cells. This heterogeneity in the timing of expression of RNA from the IE 52K gene is paralleled by the asynchronous accumulation of the protein product. We conclude that transcription of the IE-G RNA is independent of expression of the IE 52K gene and that expression of the 52K gene requires (or is prevented by) factors which do not affect accumulation of the RNA from the IE-G gene.

A cDNA library was constructed in lambda gt10 using mRNA purified from cells infected with parainfluenza virus type 2 (PIV2). Virus-specific clones were identified by screening the library with 32P-labelled cDNA probes made from randomly primed vRNA. Clones containing the haemagglutinin-neuraminidase (HN) gene were identified by sequence comparisons with known parainfluenza virus HN gene sequences. The largest HN clone isolated had a nucleic acid sequence of 2065 bp with a single long open reading frame encoding a protein of 571 amino acids. The HN protein has nine predicted glycosylation sites and an amino-terminal membrane-spanning region. The PIV2 HN protein shares 43% amino acid identity with the HN protein of simian virus 5 and 40% with mumps virus, 30% of the amino acids being common to all three viruses.

The time course of virus replication in cultures of permissive cells infected with high multiplicities of herpesvirus saimiri (HVS), a gammaherpesvirus, is protracted relative to the replication of herpes simplex virus (HSV), an alphaherpesvirus, under similar conditions. The basis for this difference was investigated by quantitative immunofluorescence microscopy exploiting monoclonal antibodies specific to the HVS 52 000 mol. wt. immediate-early polypeptide (IE 52K) and to delayed-early (DE 51K, DE 110K) and late (130K and capsid proteins) gene products to measure the timing of gene expression in individual cells of infected cultures. The timing of the transitions from IE to DE and from DE to late protein synthesis occurred at proportionately different intervals in the growth cycle of HVS, relative to that of HSV. In particular, the DE to late transition occurred relatively later in HVS infections. However, asynchrony in the events leading to the expression of the first class of virus proteins (IE 52K) was the main source of the extended course of HVS replication in populations of infected cells. This asynchrony was not modified significantly by infection at different stages of the host cell-cycle and was reduced, but not overcome, by very high applied multiplicities of infection. Double-antibody staining revealed a positive correlation between the accumulation of high concentrations of parental virus particles at perinuclear sites and early detection of HVS IE 52K gene expression. Both of these events remained sensitive to a microtubule poison (colcemid) for many hours after infection with HVS, whereas the rapid and synchronous expression of the IE 175K protein (ICP4) in HSV-1-infected cells was insensitive to post-infection exposures to this drug. We conclude that significant differences in early stages of virus entry and intracellular processing which precede immediate-early gene expression are largely responsible for differences between the replicative cycles of these representatives of gamma- and alphaherpesviruses in cultures of permissive cells.

The synthesis and accumulation of more than 30 virus-induced polypeptides was detected after infection of permissive primate cells with high multiplicities of herpesvirus saimiri. These virus-induced polypeptides had apparent mol. wt. of from 12,000 (12K) to 250K and differed in molar abundance by up to two orders of magnitude. The majority of virus-induced polypeptides satisfied multiple criteria for their virus specificity. Polypeptides of 110K, 76K, 51K and 31K to 29K were synthesized at maximum rates early in infection, but the majority of proteins were made at high rates late in infection. Virus-specific polypeptides were substrates for a number of post-synthetic modifications. The 117K, 85K, 76K, 31K and 30K polypeptides were each processed to forms with altered electrophoretic mobility. The 117K and 85K polypeptides were among the virus-specified substrates for glycosylation and virus-induced polypeptides of 59K, 51K, 30K and 26K were phosphorylated. The early 51K phosphoprotein and the 30K to 31K polypeptides were rapidly translocated to the nucleus of infected cells. The 31K polypeptide was processed to a 29K product which remained stably associated with the nuclear fraction. The 30K nuclear protein was shown to be the precursor of a 28K polypeptide which was released in a soluble form into the cytoplasmic fraction and the culture medium of cells at late times in the virus growth cycle. Many other polypeptides accumulated slowly in nuclear (e.g. 250K, 150K, 130K, 110K and 38K) or in cytoplasmic (e.g. 117K, 85K and 28K) fractions of infected cells in forms which could be differentiated by the use of detergents or differences in stability to salt extraction. The mol. wt., relative molarities and some features of the post-synthetic processing of herpesvirus saimiri polypeptides more closely resembled the properties of gene products of Epstein-Barr virus than those of herpes simplex virus.

A virus-specified polypeptide of 160,000 mol. wt. (160K) was purified more than 1000-fold from the soluble proteins present in the culture medium of cells productively infected with herpesvirus saimiri (HVS). Purified preparations of the 160K polypeptide gave rise to high titre precipitating and neutralizing antibodies in immunized rabbits, which cross-neutralized independent isolates of HVS (neutralization rate constants, k, of between 3.8 and 4.8) and specifically precipitated the 160K polypeptide from extracts of cells infected with the homologous or heterologous strains of this virus. Antiserum to the 160K polypeptide of HVS also gave low (k = 0.02) levels of neutralization of the related herpesvirus ateles. Purified preparations of the 160K polypeptide were capable of removing most or all of the neutralizing antibody sera of squirrel monkeys with naturally acquired antibodies to HVS. The 160K polypeptide was previously shown to form part of the surface of enveloped virus particles. However, we show here that the 160K polypeptide is not extensively glycosylated in infected cells. The majority of glucosamine incorporation specific to HVS-infected cells was into eight regions with apparent mol. wt. of 170K to 220K, 125K to 145K, 115K to 120K, 83K to 88K, 65K to 75K, 52K to 58K, 25K to 27K and 12.5K to 13K. It remains possible that alternative cleaved or glycosylated forms of the 160K polypeptide are also present on the virus particle or that precipitating antibody to 160K and virus-neutralizing antibodies are not identical. However, our inability to detect precipitating antibody to virus-induced proteins or glycoproteins other than the 160K polypeptide and the high titre of neutralizing antibody present in this serum, provides reasonable evidence that it is the antibodies which react with the 160K polypeptide that are responsible for virus neutralization.

Agar immunodiffusion tests demonstrated that BHK 21 cells infected with either HSV I or HSV 2 release only a few HSV-specified antigens into the extracellular fluid (infected cell released polypeptides-ICRP). Neutralization blocking experiments showed that the majority of antigens/(including the Band II common antigen) involved as target sites in antibody-mediated virus neutralization are present in the ICRP of both HSV I and HSV 2.SDS-PAGE identified six regions of virus-specified proteins in the ICRP from both HSV I- and HSV 2-infected BHK cells. All these specifically released proteins are glycosylated, although to varying degrees. The SDS-PAGE profiles of HSV I and HSV 2 ICRP are different but do show some similarities, the most notable being a highly glycosylated protein with an estimated mol. wt. of 50,000 to 54,000 in HSV I ICRP and 52,000 to 56,000 in HSV 2 ICRP. Immune precipitation demonstrated that these two proteins contain the Band II antigenic site. Similar studies showed that the major type I specific antigenic site, which is involved as a target site in the neutralization of virus infectivity, is located in the highest mol. wt. glycoprotein region of HSV I ICRP and has a similar mobility to the VP7/8 region of purified enveloped virus.

Monoclonal antibodies (MAbs) were complexed to solid matrices and used to purify virus proteins from simian virus 5 (SV5)-infected tissue culture cells, ideally in such a way as to bind equimolar amounts of antigen to antibody. The resulting solid matrix-antibody-antigen (SMAA) complexes were then used as immunogens and successfully induced specific humoral and cytotoxic T cell responses. By attaching more than one MAb to the solid matrix and using such complexes to purify the respective proteins multivalent immunization was achieved. Analysis of the cytotoxic T cell response of immunized animals indicated that both surface and internal SV5 structural proteins can act as target antigens. Immunization with SMAA complexes, in the absence of adjuvant, induced higher levels of antibody than the antigen alone precipitated on alum. However, immunization with SMAA complexes resulted in relatively less antibody being produced to the antigenic determinant through which the protein is coupled, via antibody, to the SMAA complex compared with the amount of antibody produced against other antigenic determinants on that protein. The particulate solid matrix used to form the SMAA complexes in most of the experiments was a 'fixed' and killed suspension of the Cowan A strain of Staphylococcus aureus, although preliminary results indicated that Protein A-Sepharose could also be used as a solid matrix. Prior immunization with S. aureus alone did not reduce the level of the immune response to the appropriate antigen on subsequent immunization with S. aureus-antibody-antigen complexes. In fact on immunization of mice with these complexes the level of antibody produced to the S. aureus matrix itself was less when S. aureus-antibody-antigen complexes were used as immunogens than when S. aureus or S. aureus-antibody complexes were used. Furthermore, rabbits immunized with S. aureus-mouse MAb-antigen complexes showed a vigorous immune response to the antigen.

A mouse model system has been developed to examine the ability of purified virus proteins to protect mice from infection with the paramyxovirus simian virus 5. The system is based on the infection of mouse lungs by intranasal administration of infectious virus. The relative amounts of virus proteins and nucleic acid present within infected lungs were estimated either by Western blot analysis of disrupted lung tissues or by in situ hybridization studies using cryostat sections of infected lungs. During a normal time course of infection in non-immunized mice increasing amounts of virus protein and nucleic acid were detected in the lungs until 3 days post-infection (p.i.). Thereafter the amount of virus present within the lungs remained relatively constant until 7 days p.i. when there was a rapid decrease. Cytotoxic T cells, but not neutralizing antibody, could be detected at the time when the amount of virus within the lungs was decreasing. Prior immunization of mice with solid matrix-antibody-antigen (SMAA) complexes containing either surface or internal virus structural proteins reduced the amount of virus replication within infected lungs, the greatest degree of protection being observed when nucleoprotein or matrix protein was used to immunize the mice. There was no correlation between the degree of protection observed and the level of neutralizing antibody present in immunized animals; no neutralizing antibody was detected in mice immunized with internal virus proteins even at the time of sacrifice 5 days p.i. We have previously shown that immunization of mice with SMAA complexes containing either surface or internal virus structural proteins can induce cytotoxic T cells and thus conclude that the most likely explanation for the protection observed in immunized mice is through the induction of cytotoxic T cells.

A major virus-specific immediate-early (IE) polypeptide with an apparent mol. wt. of 52 000 (52K) was synthesized immediately after the removal of a cycloheximide block applied to owl monkey kidney cell cultures at the time of infection with high multiplicities of herpesvirus saimiri (HVS). The IE 52K polypeptide was phosphorylated and accumulated rapidly and efficiently in the nucleus of infected cells. Monoclonal antibodies to IE 52K and to a major non-structural delayed-early (DE) DNA-binding protein with similar electrophoretic mobility (DE 51K) were used to show that the IE 52K protein and DE 51K proteins are antigenically distinct. The monoclonal antibody to the IE 52K protein of HVS strain 11 reacted in immune precipitation and immunofluorescence tests with polypeptides of similar mol. wt. present as nuclear antigens in cells infected with heterologous strains of HVS and herpesvirus ateles.

Two virus-induced polypeptides are preferentially released into the culture medium of owl monkey kidney cells productively infected with herpesvirus saimiri (HVS). These polypeptides have apparent mol. wt. of 160 X 10(3) and 28 X 10(3) and are the major virus-induced polypeptides in a membrane fraction from infected cells. Both these excreted polypeptides co-migrate with virus structural proteins and are selectively released from enveloped virus particles by treatment with Nonidet-P40.

Marked differences in the apparent Mrs of the HN, NP and F proteins of simian virus 5 (SV5) and parainfluenza virus type 2 (PF-2) were revealed by SDS-PAGE. To examine the antigenic relationships between SV5, PF-2 and other paramyxoviruses, monoclonal antibodies (MAbs) specific to PF-2 were isolated. These antibodies had specificities for the HN, NP and P proteins and together with 54 MAbs to SV5 were tested for their ability to react with SV5, PF-2, PF-3, mumps and measles virus proteins. Most of these MAbs (55 out of 60) reacted with homologous virus only. However, five reacted with both SV5 and PF-2. These antibodies had reactivities to the NP, M and P proteins. Furthermore, one of the antibodies with reactivity to the P protein also reacted with mumps virus. Although none of the 21 MAbs with specificities for the HN protein of either SV5 or PF-2 cross-reacted with heterologous virus, some antigenic similarities between the HN protein of SV5 and PF-2 could be detected. This was demonstrated by raising a series of polyclonal antisera to purified preparations of SV5 or PF-2 HN proteins in BALB/c mice, and testing for their ability to neutralize both SV5 and PF-2 and also to immunoprecipitate the HN proteins of these viruses. Surprisingly, while low levels of cross-neutralizing antibody could be detected in some sera (e.g. neutralization of SV5 1:1600 and of PF-2 1:80), other sera with similar neutralization titres against homologous virus failed to neutralize heterologous virus. Furthermore, only a minority of the anti-HN antisera showed any immune-precipitating activity against the heterologous HN protein.

The localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diamidino-2-phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication.

Four intertypic recombinants of herpes simplex virus have been shown to possess genetic information for functions characteristic of each of the two parental types. The functions were identified by (a) polyacrylamide gel electrophoresis of purified virus particles and of polypeptides synthesized in cells infected with the recombinants and (b) analysis of antigenic sites interacting with type specific neutralizing antibody. The analysis shows that each recombinant possess a different combination of these type specific markers. Finally we have been unable to detect recombination between herpes simplex type 1 and pseudorabies viruses.

Hybridomas secreting monoclonal antibodies to simian virus 5 (SV5) were obtained following immunization of mice with purified preparations of a human isolate (LN) of SV5. Immune precipitation studies showed that these monoclonal antibodies had specificities for the haemagglutinin-neuraminidase (HN), fusion (F), nucleo-, matrix and phospho- (P) proteins of SV5. By use of a radioimmune competition assay the monoclonal antibodies to the HN protein were assigned to four groups, members of which recognized different antigenic sites on the protein. All the anti-HN antibodies and the anti-F antibody neutralized virus infectivity. The 54 monoclonal antibodies obtained were used to determine whether there were antigenic differences between five human, two canine and one simian isolate of SV5. Although most of the monoclonal antibodies reacted with all isolates, a few did reveal antigenic differences in the HN, F and P proteins. Furthermore, analysis by SDS-PAGE showed that while the electrophoretic mobilities of most of the virus polypeptides of these isolates were similar some differences could be detected. In particular the P protein showed the most marked mobility differences between the human, canine and simian isolates. Slight differences in the mobility of the F1 glycoprotein could also be visualized.

Associations of herpesvirus saimiri-specified proteins with nuclear fractions from cultures of infected cells were probed by nuclease digestion, detergent extractions and extractions and immunofluorescence microscopy using monoclonal antibodies to virus polypeptides. Nuclease digestion selectively released delayed-early polypeptides with apparent mol. wt. of 110 000 (110K) and 51 000 (51K) from nuclei of infected cultures and the majority of each of these polypeptides partitioned with the insoluble fraction after detergent extraction of such nuclei. However, the nuclease-mediated release of both these proteins was specifically reduced when nuclei were isolated from cultures in which virus DNA synthesis had been inhibited with phosphonoacetic acid (PAA). In addition, the 110K polypeptide partitioned into the soluble fraction when nuclei from PAA-treated cultures were extracted with detergent. Immunofluorescence microscopy revealed characteristic and distinctive subnuclear localizations of the 110K and 51K polypeptides in control cultures and these patterns of subnuclear accumulations were markedly altered in cultures treated with PAA. We conclude that the DNA-binding properties of the delayed-early 110K and 51K proteins of herpesvirus saimiri previously observed in vitro are likely to reflect their functions as DNA-binding proteins in vivo.