Alteration of encephalomyocarditis virus pathogenicity due to a mutation at position 100 of VP1

Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis, myocarditis and diabetes in its natural host, the mouse. In this study, we generated four cDNA clones with a point mutation at position 100 of VP1. The amino acids isoleucine, alanine, serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis, and viable viruses were rescued. Although all mutants and wild-type viruses display different plaque morphologies, they replicate comparably in BHK-21 cells. The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice. The results showed that the isoleucine- (T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality, lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus. These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection, and its mutation alters the pathogenicity of viral infection in mice.     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.     

Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages

In response to virus infection or treatment with dsRNA, macrophages express the inducible form of cyclooxygenase-2 (COX-2) and produce proinflammatory prostaglandins. Recently, we have shown that NF-kappaB is required for encephalomyocarditis virus (EMCV)- and dsRNA-stimulated COX-2 expression in mouse macrophages. The dsRNA-dependent protein kinase R is not required for EMCV-stimulated COX-2 expression, suggesting the presence of protein kinase R-independent pathways in the regulation of this antiviral gene. In this study, the role of MAPK in the regulation of macrophage expression of cyclooxygenase-2 (COX)-2 in response to EMCV infection was examined. Treatment of mouse macrophages or RAW-264.7 cells with dsRNA or infection with EMCV stimulates the rapid activation of the MAPKs p38, JNK, and ERK. Inhibition of p38 and JNK activity results in attenuation while ERK inhibition does not modulate dsRNA- and EMCV-induced COX-2 expression and PGE2 production by macrophages. JNK and p38 appear to selectively regulate COX-2 expression, as inhibition of either kinase fails to prevent dsRNA- or EMCV-stimulated inducible NO synthase expression by macrophages. Using macrophages isolated from TLR3-deficient mice, we show that p38 and JNK activation and COX-2 expression in response to EMCV or poly(IC) does not require the presence the dsRNA receptor TLR3. These findings support a role for p38 and JNK in the selective regulation of COX-2 expression by macrophages in response to virus infection.     

Down-regulation of p53 by double-stranded RNA modulates the antiviral response

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G(1) arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G(1) arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.     

Rapamycin and Wortmannin Enhance Replication of a Defective Encephalomyocarditis Virus

Inhibitors of the phosphatidylinositol 3-kinase (PI3 kinase)–FKBP-rapamycin-associated protein (FRAP) pathway, such as rapamycin and wortmannin, induce dephosphorylation and activation of the suppressor of cap-dependent translation, 4E-BP1. Encephalomyocarditis virus (EMCV) infection leads to activation of 4E-BP1 at the time of host translation shutoff. Consistent with these data, rapamycin mildly enhances the synthesis of viral proteins and the shutoff of host cell protein synthesis after EMCV infection. In this study, two defective EMCV strains were generated by deleting portions of the 2A coding region of an infectious cDNA clone. These deletions dramatically decreased the efficiency of viral protein synthesis and abolished the virus-induced shutoff of host translation after infection of BHK-21 cells. Both translation and processing of the P1-2A capsid precursor polypeptide are impaired by the deletions in 2A. The translation and yield of mutant viruses were increased significantly by the presence of rapamycin and wortmannin during infection. Thus, inhibition of the PI3 kinase-FRAP signaling pathway partly complements mutations in 2A protein and reverses a slow-virus phenotype.     

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.     

Catalytic properties of a human cytomegalovirus-induced protein kinase

Human cytomegalovirus, a DNA virus whose genome contains a fragment of transforming DNA, induces a threonine-serine protein kinase having a molecular mass of 68 kDa (p68). p68 was extracted from cells 96-144 h after infection, and immunoprecipitated with a monoclonal antibody (F6b). Antibody-enzyme complexes were immobilized on heat/formaldehyde-inactivated Staphylococcus aureus. The best substrates for p68 were acidic proteins, phosvitin and casein. Glycogen synthase, phosphorylase alpha and histones were phosphorylated at rates not higher than 1-4% that obtained with phosvitin as substrate. ATP and GTP were equally good substrates of p68. p68 is able to autophosphorylate at the same residues (i.e. threonine and serine) as the protein substrates. Autophosphorylation does not seem to represent an intermediate in substrate phosphorylation. The protein kinase activity of p68 was not enhanced by cAMP, calcium ions, or polyamines like spermine or spermidine. Only at low Mg2+ concentration spermine enhanced by 68% the rate of casein phosphorylation. Heparin, a potent inhibitor of casein kinase II, inhibits p68 activity too, but ten-times higher concentrations were required for the same degree of inhibition. Quercetin, a bioflavonoid, acts as a strong inhibitor of p68 protein kinase activity. The inhibitory effect of quercetin was competitive towards the nucleotide substrate (Ki = 2.8 microM), and non-competitive towards the protein substrate (Ki = 15 microM).     

NF-κB-Mediated Inhibition of Apoptosis Is Required for Encephalomyocarditis Virus Virulence: a Mechanism of Resistance in p50 Knockout Mice

Apoptosis is a central host defense mechanism to eliminate virus-infected cells. Activation of NF-κB suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine encephalomyocarditis virus (EMCV) infection in mice and cell lines defective in NF-κB1 (p50) signaling. As previously reported, we find that all p50 knockout (p50 −/−) mice survive an EMCV infection that readily kills normal mice. By introducing the p50 mutation into interferon (IFN) type I receptor knockout (IFNRI −/−) mice, we find that this resistance is not mediated by IFN-β as previously thought. While no IFNRI −/− mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals’ ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 −/−, and p65 −/− embryos, we found that NF-κB is not required for the replication cycle of EMCV. However, during these experiments we observed that p50 −/− and p65 −/− MEF infected with EMCV undergo enhanced, premature cytotoxicity. Upon examination of this cell death, we found that EMCV infection induced both plasma membrane and nuclear changes typical of apoptosis in all cell lines. These apoptotic processes occurred in an accelerated and pronounced way in the NF-κB-defective cells, as soon as 6 h after infection, when virus is beginning to be released. Previously, only the RelA (p65) subunit of NF-κB has been shown to play a role in suppressing apoptosis. In our studies, we find that p50 is equally important in suppressing apoptosis during EMCV infection. Additionally, we show that suppression of apoptosis by NF-κB1 is required for EMCV virulence in vivo. The attenuation in p50 −/− mice can be explained by rapid apoptosis of infected cells which allows host phagocytes to clear infected cells before the viral burst leading to a reduction of the viral burden and survival of the mice.     

Aberrant protein phosphorylation at tyrosine is responsible for the growth-inhibitory action of pp60v-src expressed in the yeast Saccharomyces cerevisiae.

Expression of pp60v-src, the transforming protein of Rous sarcoma virus, arrests the growth of the yeast Saccharomyces cerevisiae. To determine the basis of this growth arrest, yeast strains were constructed that expressed either wild-type v-src or various mutant v-src genes under the control of the galactose-inducible, glucose repressible GAL1 promoter. When shifted to galactose medium, cells expressing wild-type v-src ceased growth immediately and lost viability, whereas cells expressing a catalytically inactive mutant (K295M) continued to grow normally, indicating that the kinase activity of pp60v-src is required for its growth inhibitory effect. Mutants of v-src altered in the SH2/SH3 domain (XD4, XD6, SPX1, and SHX13) and a mutant lacking a functional N-terminal myristoylation signal (MM4) caused only a partial inhibition of growth, indicating that complete growth inhibition requires either targeting of the active kinase or binding of the kinase to phosphorylated substrates, or both. Cells arrested by v-src expression displayed aberrant microtubule structures, alterations in DNA content and elevated p34CDC28 kinase activity. Immunoblotting with antiphosphotyrosine antibody showed that many yeast proteins, including the p34CDC28 kinase, became phosphorylated at tyrosine in cells expressing v-src. Both the growth inhibition and the tyrosine-specific protein phosphorylation observed following v-src expression were reversed by co-expression of a mammalian phosphotyrosine-specific phosphoprotein phosphatase (PTP1B). However a v-src mutant with a small insertion in the catalytic domain (SRX5) had the same lethal effect as wild-type v-src, yet induced only very low levels of protein-tyrosine phosphorylation. These results indicate that inappropriate phosphorylation at tyrosine is the primary cause of the lethal effect of pp60v-src expression but suggest that only a limited subset of the phosphorylated proteins are involved in this effect.     

Studies on the role of the 2''-5''-oligoadenylate synthetase-RNase L pathway in beta interferon-mediated inhibition of encephalomyocarditis virus replication.

Interferons inhibit the replication of vesicular stomatitis virus (VSV), but not of encephalomyocarditis virus (EMCV), in mouse JLSV-11 cells. We report the isolation of clonal derivatives from this cell line in which the replication of both viruses is impaired by interferons. These clones were selected from the parental line by virtue of their rescue by interferon treatment from the cytopathic effects of EMCV infection. In one such clone, RK8, the replication of VSV and EMCV and the production of resident murine leukemia virus were inhibited by interferon. On the other hand, in clone RK6, which was isolated without any selection, the replication of VSV, but not of EMCV, was impaired by interferons. The levels of 2'-5'-oligoadenylate synthetase mRNA and enzyme activity were similarly elevated upon interferon treatment in the two clones. However, the level of RNase L, as determined by binding and cross-linking of a radiolabeled 2'-5'-oligoadenylate derivative, was much lower in RK6 cells than in RK8 cells. In accord with this observation, the introduction of 2'-5'-oligoadenylates into cells inhibited protein synthesis much less strongly in RK6 cells than in RK8 cells. These results are consistent with the notion that the 2'-5'-oligoadenylate-dependent RNase L may be a mediator of the inhibition of EMCV replication by interferons.     

Encephalomyocarditis virus 2A protein is required for viral pathogenesis and inhibition of apoptosis

The encephalomyocarditis virus (EMCV), a Picornaviridae virus, has a wide host spectrum and can cause various diseases. EMCV virulence factors, however, are as yet ill defined. Here, we demonstrate that the EMCV 2A protein is essential for the pathogenesis of EMCV. Infection of mice with the B279/95 strain of EMCV resulted in acute fatal disease, while the clone C9, derived by serial in vitro passage of the B279/95 strain, was avirulent. C9 harbored a large deletion in the gene encoding the 2A protein. This deletion was incorporated into the cDNA of a pathogenic EMCV1.26 strain. The new virus, EMCV1.26Δ2A, was capable of replicating in vitro, albeit more slowly than EMCV1.26. Only mice inoculated with EMCV1.26 triggered death within a few days. Mice infected with EMCV1.26Δ2A did not exhibit clinical signs, and histopathological analyses showed no damage in the central nervous system, unlike EMCV1.26-infected mice. In vitro, EMCV1.26Δ2A presented a defect in viral particle release correlating with prolonged cell viability. Unlike EMCV1.26, which induced cytopathic cell death, EMCV1.26Δ2A induced apoptosis via caspase 3 activation. This strongly suggests that the 2A protein is required for inhibition of apoptosis during EMCV infection. All together, our data indicate that the EMCV 2A protein is important for the virus in counteracting host defenses, since Δ2A viruses were no longer pathogenic and were unable to inhibit apoptosis in vitro.     

Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae.

Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae. The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected. Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone. Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry. p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs. These results suggest that wild-type p53 has a growth-inhibitory activity in S. cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.     

Regulation of p90RSK phosphorylation by SARS-CoV infection in Vero E6 cells

The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

The cellular 68,000-Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation.

We investigated the possible translational regulatory roles played by the interferon-induced, double-stranded-RNA-activated protein kinase (P68) and its natural substrate, eucaryotic initiation factor 2 (eIF-2), in poliovirus-infected cells. We demonstrated that protein kinase P68 was both highly autophosphorylated and activated during poliovirus infection. In accordance with these results, immunoprecipitation analysis revealed that phosphorylation of the endogenous eIF-2 alpha subunit also increased in poliovirus-infected cells. We found that double-stranded RNA synthesized during infection likely induced the high levels of P68 autophosphorylation. To determine whether the increase in kinase activity also could be attributed to induction of P68 synthesis, physical levels of protein kinase were measured. It was unexpectedly found that P68 protein levels did not increase but rather dramatically declined in poliovirus-infected cells. Pulse-chase experiments confirmed that the protein kinase was significantly degraded during virus infection. We corroborated our in vivo observations by developing an in vitro assay for P68 degradation using cell extracts. The possible consequences of P68 degradation and increased eIF-2 alpha phosphorylation for protein synthesis regulation in poliovirus-infected cells are discussed.     

Regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded RNA and viral infection

In this study the regulation of macrophage expression of cyclooxygenase-2 (COX-2) in response to dsRNA and virus infection was examined. Treatment of RAW 264.7 macrophages with dsRNA results in COX-2 mRNA accumulation and protein expression and the production of PGE(2). Similar to dsRNA, encephalomyocarditis virus (EMCV) infection of RAW 264.7 cells stimulates COX-2 expression and PGE(2) accumulation. The dsRNA-dependent protein kinase (PKR), which has been shown to participate in the regulation of gene expression in response to dsRNA and virus infection, does not appear to participate in the regulation of COX-2 expression by macrophages. Expression of dominant negative mutants of PKR in RAW 264.7 cells fails to attenuate dsRNA- and EMCV-induced COX-2 expression or PGE(2) production. Furthermore, dsRNA and EMCV stimulate COX-2 expression and PGE(2) accumulation to similar levels in macrophages isolated from wild-type and PKR-deficient mice. Recently, a novel PKR-independent role for the calcium-independent phospholipase A(2) (iPLA(2)) in the regulation of inducible NO synthase expression by macrophages in response to virus infection has been identified. The selective iPLA(2) suicide substrate inhibitor bromoenol lactone prevents dsRNA- and EMCV-stimulated inducible NO synthase expression; however, bromoenol lactone does not attenuate dsRNA- or EMCV-induced COX-2 expression by macrophages. In contrast, inhibition of NF-kappaB activation prevents dsRNA-stimulated COX-2 expression and PGE(2) accumulation by macrophages. These findings indicate that virus infection and treatment with dsRNA stimulate COX-2 expression by a mechanism that requires the activation of NF-kappaB and that is independent of PKR or iPLA(2) activation.     

CD1d mediates T-cell-dependent resistance to secondary infection with encephalomyocarditis virus (EMCV) in vitro and immune response to EMCV infection in vivo

The innate and adaptive immune responses have evolved distinct strategies for controlling different viral pathogens. Encephalomyocarditis virus (EMCV) is a picornavirus that can cause paralysis, diabetes, and myocarditis within days of infection. The optimal innate immune response against EMCV in vivo requires CD1d. Interaction of antigen-presenting cell CD1d with distinct natural killer T-cell ("NKT") populations can induce rapid gamma interferon (IFN-gamma) production and NK-cell activation. The T-cell response of CD1d-deficient mice (lacking all NKT cells) against acute EMCV infection was further studied in vitro and in vivo. EMCV persisted at higher levels in CD1d-knockout (KO) splenocyte cultures infected in vitro. Furthermore, optimal resistance to repeat cycles of EMCV infection in vitro was also shown to depend on CD1d. However, this was not reflected in the relative levels of NK-cell activation but rather by the responses of both CD4(+) and CD8(+) T-cell populations. Repeated EMCV infection in vitro induced less IFN-gamma and alpha interferon (IFN-alpha) from CD1d-deficient splenocytes than with the wild type. Furthermore, the level of EMCV replication in wild-type splenocytes was markedly and specifically increased by addition of blocking anti-CD1d antibody. Depletion experiments demonstrated that dendritic cells contributed less than the combination of NK and NKT cells to anti-EMCV responses and that none of these cell types was the main source of IFN-alpha. Finally, EMCV infection in vivo produced higher levels of viremia in CD1d-KO mice than in wild-type animals, coupled with significantly less lymphocyte activation and IFN-alpha production. These results point to the existence of a previously unrecognized mechanism of rapid CD1d-dependent stimulation of the antiviral adaptive cellular immune response.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

Two point mutations in the transmembrane domain of P68gag-ros inactive its transforming activity and cause a delay in membrane association.

The transforming protein of the avian sarcoma virus UR2 is a 68-kDa transmembrane tyrosine protein kinase. We examined the relationship between membrane localization and transforming activity of P68 by changing Val-168-Val-169 in its hydrophobic domain into Asp-168-Glu-169. The resulting transmembrane (TM) mutant (P68TM) lost transforming activity toward chicken embryo fibroblasts (CEF). We found that the mutant protein was expressed and rapidly degraded into a smaller form which was still membrane associated and kinase active. The instability of the TM mutant protein is a phenomenon only manifested in CEF, because the same mutant protein was expressed with efficiency and stability similar to those of the wild-type protein in a transient expression system in COS cells. However, there are several differences between the wild-type and the TM mutant proteins in COS cells. The wild-type protein is more heavily phosphorylated and associated with membrane fractions in a cotranslational manner. It is enzymatically active when recovered from membrane fractions. The TM mutant protein is less phosphorylated, more labile toward protease degradation, and delayed in membrane association, with a lag period of 30 min or longer, and has little kinase activity when recovered from membrane fractions. Most of the kinase-active TM mutant protein was found in the cytosol fractions. Despite the delay, most of the TM protein in COS cells was found to be membrane associated, and its orientation on the cell surface was similar to that of the wild-type protein. It is probable that loss of the CEF-transforming activity of the TM mutant protein is due to its susceptibility to protease degradation resulting from improper membrane association of the newly synthesized product. The differences in the kinetics of membrane association and the distribution of kinase activity in COS cells might not be directly applicable in explaining the inability of the TM mutant to transform CEF but are intriguing as regards protein biosynthesis and translocation. The difference between CEF and COS cells implies that different factors or pathways are involved in the biosynthesis and processing of the TM mutant protein in these two cellular environments. Changes of P68TM in the kinetics of membrane association indicate that the transmembrane domain of ros, besides functioning as a membrane anchor, also plays a role in directing initial membrane association.