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Unlabelled: Stress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition., Importance: Our previous work indicates that stress granules have antiviral activity and mediate innate immunity through functions of G3BP1; however, the mechanistic details of these functions were not resolved. We show that much of the antiviral activity of stress granules is contingent on the function of PKR in a complex with G3BP1 and Caprin1. The PKR-G3BP1-Caprin1 complex undergoes dynamic transitioning within and outside stress granules to accomplish PKR activation and translational repression. This mechanism appears to function distinctly from canonical pattern recognition of double-stranded RNA by PKR. Therefore, this mechanism bridges the stress response with innate immunity, allowing the cell to respond to many cellular stressors and amplify the pathogen pattern recognition systems of innate immunity., (Copyright © 2015 Reineke et al.)

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

Although the ability of serum-neutralizing antibodies to protect against picornavirus infection is well established, the contribution of cell-mediated immunity to protection is uncertain. Using major histocompatibility complex class II-deficient (RHAbeta-/-) mice, which are unable to mediate CD4(+) T-lymphocyte-dependent humoral responses, we demonstrated antibody-independent protection against lethal encephalomyocarditis virus (EMCV) infection in the natural host. The majority of RHAbeta-/- mice inoculated with 10(4) PFU of attenuated Mengo virus (vMC24) resolved infection and were resistant to lethal challenge with the highly virulent, serotypically identical cardiovirus, EMCV. Protection in these mice was in the absence of detectable serum-neutralizing antibodies. Depletion of CD8(+) T lymphocytes prior to lethal EMCV challenge ablated protection in vMC24-immunized RHAbeta-/- mice. The CD8(+) T-lymphocyte-dependent protection observed in vivo may, in part, be the result of cytotoxic T-lymphocyte (CTL) activity, as CD8(+) T splenocytes exhibited in vitro cytolysis of EMCV-infected targets. The existence of virus-specific CD8(+) T-lymphocyte memory in these mice was demonstrated by increased expression of cell surface activation markers CD25, CD69, CD71, and CTLA-4 following antigen-specific reactivation in vitro. Although recall response in vMC24-immunized RHAbeta-/- mice was intact and effectual shortly after immunization, protection abated over time, as only 3 of 10 vMC24-immunized RHAbeta-/- mice survived when rechallenged 90 days later. The present study demonstrating CD8(+) T-lymphocyte-dependent protection in the absence of serum-neutralizing antibodies, coupled with our previous results indicating that vMC24-specific CD4(+) T lymphocytes confer protection against lethal EMCV in the absence of prophylactic antibodies, suggests the existence of nonhumoral protective mechanisms against picornavirus infections.

C57BL/6 mice develop a virus-specific cytotoxic T-lymphocyte (CTL) response after intraperitoneal inoculation with either the DA strain of Theiler's virus or Mengo virus, two members of the Cardiovirus genus. These CTLs contribute to viral clearance in the case of Theiler's virus but do not protect the mice from the fatal encephalomyelitis caused by Mengo virus. In this study we show that DA and Mengo virus-induced CTLs are cross-reactive. The cross-reactivity is due to a conserved, H-2Db-restricted epitope located between amino acid residues 122 and 130 of the VP2 capsid protein (VP2(122-130)). This epitope is immunodominant in C57BL/6 mice infected with Theiler's virus. The VP2(122-130) epitope, initially identified for Mengo virus, is the first CTL epitope described for Theiler's virus.

The development of safe and effective vaccines remains a major goal in the prevention, and perhaps treatment, of infectious diseases. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of the immune response. We originally demonstrated that two virus-specific cytotoxic T-lymphocyte (CTL) epitopes, from the same virus but presented by different major histocompatibility complex alleles, when linked in tandem as minigenes in a recombinant vaccinia virus, could confer complete protection against subsequent viral challenge. In the study, we extended this approach, which we termed string of beads, expanding the immunogenic scope in two ways: first, by introduction of T helper (Th) and B-cell (antibody) epitopes alongside CTL epitopes and second, by including immunogenic sequences from a variety of infectious agents, five viruses and one bacterium. The vaccine (VV-sv) comprises CTL epitopes from Sendai virus, respiratory syncytial virus, and lymphocytic choriomeningitis virus (LCMV); Th epitopes from vesicular stomatitis virus and Mycobacterium tuberculosis; and an antibody epitope from mengovirus. The construct contains a single start codon, and the epitopes are linked directly, without intervening spacer amino acids. There was some concern that the combination of several normally immunodominant epitopes might result in a new hierarchy of dominance, in which certain epitopes predominated and others exhibited reduced immunogenicity. However we show that when analyzed in tissue culture and in vivo, all six epitopes are expressed. CTL and Th cells are induced in vivo, along with neutralizing antibody. The induced immunity is biologically relevant: after VV-sv immunization, the antimengovirus antibody confers protection against mengovirus challenge. Similarly, CTL induced by the LCMV epitope protected mice against challenge with this agent. Thus, a polyvalent, minigene-based vaccine can simultaneously induce several classes of immune response and thereby can confer protection against diverse pathogens.

Several features make Mengo virus an excellent candidate for use as a vaccine vector. The virus has a wide host range, including rodents, pigs, monkeys, and most likely humans, and expresses its genome exclusively in the cytoplasm of the infected cell. Stable attenuated strains exist which are deleted for part of the 5' noncoding region of the genome. Here we report an attenuated Mengo virus recombinant, vLCMG4, that encodes an immunodominant cytotoxic T-lymphocyte epitope of the lymphocytic choriomeningitis virus (LCMV) nucleo-protein. vLCMG4 induced protective immunity against lethal LCMV infection after a single, low-dose immunization in BALB/c mice and elicited an LCMV-specific CD8+ cytotoxic T lymphocyte response. This demonstrates the potential of recombinant Mengo virus vaccines to confer protection against infectious diseases by the induction of cellular immune responses.

UV irradiation of purified mengovirus resulted in a very rapid inactivation of the infectivity of the virions (D(37) [37% survival dose] = 700 ergs/mm(2)) which correlated in time with the formation of uracil dimers in the viral RNA. During the first 2 min of irradiation, an average of 1.7 uracil dimers were formed per PFU of virus inactivated. Hemagglutination activity of the virions began to decrease only after a lag period of about 5 min and at a much lower rate (D(37) = 84,000 ergs/mm(2)). This decrease coincided in time with the appearance of altered proteins in the capsid and a structural change in the capsid. Although 10- to 20-min irradiated virions appeared intact in the electron microscope and sedimented at 150S in sucrose density gradients, the RNA of the virions became accessible to RNase and extractable by low concentrations of sodium dodecyl sulfate, and the virions broke down upon equilibrium centrifugation in CsCl gradients. During longer periods of irradiation (30 to 60 min), a progressively greater proportion of the virions were converted to 14S protein particles and 80S ribonucleoprotein particles composed of intact viral RNA and about 30% of the capsid proteins, alpha, beta, and gamma. Empty capsids were not detectable at any time during 60 min of irradiation, by which time disruption of the virions was complete. Irradiation of complete virions also resulted in an increased sedimentation rate of the viral RNA and in the covalent linkage to the viral RNA of about 1% of the total capsid protein in the form of heterogeneous low-molecular-weight polypeptides. The two observations seem to be causally related, since irradiation of isolated viral RNA did not result in an increase in sedimentation rate of the RNA, even though uracil dimer formation in viral RNA occurred at about the same rate and to the same extent whether intact virions or viral RNA were irradiated.

In mice given single intraperitoneal doses of interferon, serum interferon levels peaked at 1 h postinjection and were reduced to zero at about 8 h. The interferon concentrations in spleen, liver, and lungs were about 100-fold higher than could be expected from the amount of serum contained in these organs. In the brain only low levels of antiviral activity were detected. In mice infected intraperitoneally with Mengo virus, viral replication in the brain occurred around day 4 and was accompanied by the appearance of large amounts of interferon (approximately 10(3.25) U/g). This was preceded, however, by viral replication in the spleen and by the appearance of modest amounts of interferon in spleen and serum. In these mice protection could be obtained with relatively small doses of interferon, provided protection could be obtained with relatively small doses of interferon, provided they were given before the time of maximal levels of endogenous serum interferon. In mice infected intranasally with vesicular stomatitis virus, virus replication in the brain started within 24 to 48 h and increased with time; also, small amounts of interferon (10(2) to 10(2.5) U/g) were already detectable on days 1 and 2. The major peak of virus replication in the brain occurred on days 5 to 6 and was accompanied by the appearance of large amounts of interferon (approximately 10(3.25) U/g). In this model early treatment with interferon also provided protection, but only if given in larger doses than in the Mengo virus system. Athymic (nu/nu) mice developed a chronic systemic infection when inoculated with a demotropic strain of vaccinia virus. No interferon was detected in sera, livers, spleens, or lungs of these animals; some mice had low levels of interferon-like antiviral activity in the brain, but no attempt was made to characterize this material. Daily administration of large doses of interferon failed to exert an effect on the development of this chronic disease. Yet, normal (NMRI) mice were protected against acute infection with dermotropic or neurotropic strains of vaccinia virus, and athymic mice were partially protected against acute lethal infection with neurotropic vaccinia virus.

Mengovirus 14S subviral protein particles generated in infected L cells and in a cell-free translation system primed with mengovirus RNA were purified by sucrose gradient centrifugation and immunoaffinity chromatography. The preparations from both sources contained essentially pure proteins epsilon, alpha, and gamma, as was demonstrated in terms of virus-specific proteins (by autoradiography) and total protein content (by silver staining of sodium dodecyl sulfate-polyacrylamide electrophoresis gels). These purified proteins sedimented as discrete particles at the 14S position when recentrifuged in sucrose gradients. Although their assembly properties have not yet been studied in detail, preliminary results indicate that during incubation with virion RNA the 14S particles purified from infected cells can form a structure cosedimenting with mature mengovirus.

The present study was undertaken to examine possible correlations between long-term protection of mice treated with polycarboxylates and infected with vaccinia virus, and the presence of subdetectable amounts of interferon in the tissues, as determined by tissue or organ resistance to replication of viruses. After a single dose of polycarboxylate, splenic resistance to virus replication could be detected. It persisted for 7 to 14 days, but no attempt was made to prove that it was due to subdetectable amounts of interferon. Whole-animal protection lasted longer than splenic resistance. Moreover, when different polycarboxylates and different virus strains were used, patterns of early protection were not correlated with those of splenic resistance. These data, as others presented earlier, suggest that, in addition to interferon, other antiviral mechanisms are stimulated by polycarboxylates.