EMC virus

Procedure for production of herpes simplex, pseudorabies or EMC virus... 

Set up 20 X 50 mm dishes with 2-4 X 106BHK21 03 (EMC virus) or BSC1 (SV40) cells per dish and grow for 24 h at 37°C to obtain confluent monolayers. 

Kaufman, R. J., Davies, M. V., Wasley, L. C. and Michnick, D. (1991). Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res. 19, 4485-4490. 

We have prepared several different molecular fractions of pyran, poly(acrylic acid-co-maleic acid) PAAMA, poly(maleic acid) PMA, and poly(acrylic acid-co-3,6-endoxo-l,2,3,6-tetrahydrophtha-lic acid) BCEP and evaluated their activity against Lewis lung carcinoma (Table I) andencephalomyocarditls (EMC) virus (Table II). Table I. Inhibition of Tumor Size with Polymer Molecular Weight... 

Tnorpholonluin quaternary compounds have been shown to be active In mice Infected with mouse hepatitis and herpes simplex viruses.vfe n cell culture, N -furfurylblquanlde was active against a variety of RNA viruses.— A lengthy report on the effect of salicylates on EMC virus In cell culture Is not completely convlnclngV Certaln derivatives of it-oxo-5 thlazoll-dlne are claimed to have a wide antiviral spectrum but only data for poliovirus inhibition are presented.V ... 

There have been numerous other host resistance models used for immunotox-icity testing and these have been discussed and reviewed by Burleson and Burleson (2007). The Candida albicans fungal model (Herzyk et al., 2001) is another important model and may be used instead of or in addition to the Listeria monocytogenes model. Candida-specr c IgG and cytokines may also be quantified. Other bacterial host resistance models include Pseudomonas aeruginosa, Escherichia colt, Staphylococcus aureus, and Klebsiella pneumoniae, parasitic host resistance models including malaria and Trichinella spiralis, and viral host resistance models including encephalomyocarditis (EMC) virus, vesicular stomatitis virus (VSV), and reovirus. 

Encephalomyocarditis (EMC) virus, strain MM. A virus reduction of about 25% was seen. This is considered to be a moderate antivirial response. 

Encephalomyocarditis (EMC) virus RNA Mouse (usually grown in Krebs ascites cells) Virus specific protein (C) 100 /ig/ml Mathews and Korner (1970a) Smith el of. (1970) Dobos et of. (1971)... 

In contrast to the results discussed above, there are a few examples in the literature in which the specificity of a tissue for mRNA translation is apparently quite clearly demonstrated though by no means fully understood. One of these is the inability of reticulocyte lysates to translate EMC virus RNA without the addition of a post-ribosomal supernatant fraction from Krebs ascites eells (Mathews and Korner, 1970b Mathews, 1970). The ascites fraction contains both protein and RNA components, but these have not been further characterized. 

Information regarding the assembly of picomaviruses has been obtained largely from the characterization of subviral particles found in infected cells. In the case of poliovirus these include 6 and 15-14 particles, 80 "procapsids and 125 "provirions (68, 69, 70). The cardioviruses do not produce stable procapsids or provirions in vivo however, 15 and 14 particles have been identified in EMC virus-infected cells (7I), and recently a 5OS particle has been isolated from Mengo virus-infected cells (75T Relationships among... 

Althou most experiments clearly indicate that early after infection ribosomal EEA is the first species of ENA to be inhibited, experiments using isolated nuclei (21, 22) from mengovirus infected L-cells and EMC virus infected mouse plasmacytoma cells indicate that the polymerase II activity (responsible for heterogeneous nuclear ENA and mENA synthesis) is inhibited 1-2 hours before ENA polymerase I and III activities (responsible for rENA and 48 and 58 ENA synthesis, respectively). No difference in activity and relative proportions of the three ENA polymerases, however was found after infection using solubilized enzymes assayed in the presence of exogeneous DNA as template (2l). It is assumed that the inhibition in whole cells results from an initiation defect, since these measurements using nuclei and solubilized enzymes do not measure true initiation. 0 he explanation as to why polymerase II should be inhibited before polymerase I when rENA synthesis is clearly the first to be inhibited in whole cells remains to be worked out. 

Another theory to explain the shut-off, proposed by Cooper et al. (4Q) involves the synthesis of viral proteins which have an affinity for the small ribosomal subunit and the 5 end of viral mENA. The viral protein would repress the synthesis of cellular proteins by combining with the 4OS subunit, thereby blocking its link with host mENA. By also binding to the 5 end of viral ENA, the proteins would facilitate the attachment of viral ENA to the 40s subunit and increase the translation of viral ENA. Viral proteins have been found to co-sediment with the 4OS subunits of HeLa cells infected with poliovirus (49)> of Ehrlich ascites tumor cells infected with EMC virus (50) and of L-cells infected with mengovirus (5 ) poliovirus infected cells, the viral proteins co-sedimenting with ribosomes were identified as VPO, VP1 and VP3, all structural proteins (49) Both structural and non-structural proteins were found associated with ribosomes from EMC... 

Another way in which picomaviruses could inhibit cellular protein synthesis would be to inactivate an initiation factor needed for cellular, but not viral, mENA translation. If this were the case one might expect to find a decreased capacity of extracts from infected cells to initiate translation of cellular mENAs compared to extracts from uninfected cells. Such studies have yielded a variety of results. In some laboratories no differences in activity were detected between extracts from xininfected cells and from EMC infected plasmacytoma cells or mengovirus infected Ehrlich ascites tumor cells (26, 44) In one laboratory the ability of extracts from infected cells to translate exogenously added encephalomyocarditis (EMC) virus ENA and total Krebs II ascites cell mENA was markedly diminished, but no evidence for the selective inhibition of translation of host cell mENA was obtained (52). 

GOLINI, P., TEACH, S., LAWRENCE, C. and THACH, R. Regulation of protein synthesis in EMC virus-infected cells. In Animal... 

Cell extracts prepared at different times after infection of HeLa cells with EMC virus have been used to study the mechanism of shut-off. (These studies were made possible by the observation of Weber al. (11) that HeLa cell extracts supplemented with haemin and other components necessary for m vitro protein s3nithesis were active in initiation. 

Inhibition of protein synthesis in intact HeLa cells infected with EMC virus was observed at the same time that viral RNA synthesis reached its peak (Figure l). The shut-off of host protein synthesis, however, preceded this general inhibition of protein synthesis (see below). When cell extracts were prepared at different times after infection and tested for endogenous protein synthesis, a progressive loss of activity with time of infection was observed. A similar result was obtained in L-cells infected with EMC (Figure 2). 

That the gene order and cleavage pattern of a rhinovirus is similar to that of poliovirus and cardiovirus was first shown by McLean and Rueckert (8) who found that rhinoviruses 1A and 2, like poliovirus and EMC virus, redirect host-protein synthesis to that of the virus and produce a cleavage pattern homologous to that of EMC virus. These results were confirmed by Butterworth (9) who carried out a usefiil comparative study on all three of these viruses (Figure I). 

Stoichiometric analysis of chains in EMC virus-infected HeLa cells showed that, after correction for loss due to cleavage, the A,... 

Figure 6. Effect of pactamycin on the distribution of radioactivity incorporated into the primary products of EMC-virus directed protein synthesis. At 5 iu 49 niin post infection, an infected cell suspension was exposed to pactamycin. Six min thereafter, samples were removed, pulse labeled with a (h)-amino acid mixture and electrophoresed on SDS-polyaciylamide gels (see ref. 57) Control, 6 min pulse (solid line) pactamycin, 6 min delay, 6 min pulse (dotted line). From Butterworth and Rueckert (57) ...

EigTire 7 Effect of pactamycin on the distribution of radioactivity incorporated into the stable EMC virus polypeptides. 

If the entire picornavirus genome is translated into a single giant polyprotein which is cleaved to form the easily detectable primary proteins, A, F and C, then these proteins and their cleavage products should appear in a molar ratio of 1 1 1. If one calculates the molar ratios of the primary products as mentioned earlier, the results shown in Table 2 are obtained for mengovirus f22, 25), EMC virus (25) human rhinovirus 1A (25) and poliovirus (59) The A F C ratio for EMC virus (25) was found to be close to the theoretical value of 1 1 1. That for HRV-1A was also close to the theoretical value, 0.85 1.00 0.44> though the value for C was low. In contrast, the ratios for mengovirus (determined in two independent laboratories) and poliovirus deviated from the expected values. The capsid proteins appeared to be overproduced nearly by a factor of 2. 

The time course of induction of virus-specific protease activity in poliovirus and EMC virus-infected cells has been examined (55> 57) Th rate of new enzyme production and the amount is positively correlated with the quantity of infecting... 

Pelham (20) has provided independent evidence for a viral contribution to the proteolysis of EMC virus precursor proteins. 

In addition, a proteolytic activity was detected apparently synthesized in the cell-free system, which cleaved the coat precursor polypeptide of the virus into fragments matching in size the virus coat proteins produced in infected cells. In agreement with the results discussed for poliovirus, the results tend to rule out an autoproteolytic activity associated with the coat proteins. This is in conflict with an earlier study on EMC virus proteolysis (37) > in which coat polypeptide gamma, co-purified with an endo proteolytic activity. 

Inhibitors of well known metallo-, sulfhydi yl and serine proteases block viral proteolysis in infected cells. The use of iodoacetamide (lAM) to stabilize poliovirus and foot-and-mouth virus precursors has been described (36, 52). The preciirsors were subsequently cleaved in vitro to yield normal-sized products (36)1 and polio RHA replicase activity was stabilized in cell-free extracts of lAM-treated cells (55) H-ethyl maleimide inactivates an EMC virus-specific protease in reticulocyte lysates (20). These results could signify that a sulfhydryl-type protease, e.g., cathepsin B, is required for some of the cleavage reactions. However, when such compounds are added to cells, numerous proteins react, and there is substantial inhibition of protein synthesis. Energy metabolism in such cells is depressed, and ATP-reqiiiring proteolytic reactions blocked. 

A diagram of the overlap in amino sequences of the major translation products (l-Vl) of EMC virus RNA by a Krebs or Ehrlich ascites cell-free system is shown below. The data are from Hunt (1 5) ... 

However, pulse-chase experiments in vitro, or the addition to extracts of inhibitors of proteolytic enzymes, have failed to provide any indication that cleavage or correct processing of large polypeptides occurred. However, one consistent finding is that uninfected cell extracts translate EMC virus ENA to produce a major polypeptide approximately 20,000 daltons larger than is produced by translation of the same ENA in extracts from infected cells (5, 12). The smaller product from infected cells is the same size as the... 

More recently, EMC virus RNA was translated in a rabbit reticulocyte lysate made mRNA-dependent by prior treatment with micrococcal nuclease (1 )- This system has been shown to maintain a high elongation rate, to be relatively free of ribonuclease activity, and to avoid premature termination on other mRNAs (I8). 

In contrast to the results obtained previously with other translation systems, EMC virus RNA appeared to be translated efficiently and completely. Additional mouse liver tRNA was added to the treated reticulocyte lysate. The absence of products arising from premature termination indicate that most, if not all, ribosomes completed translation of the RNA. In addition, cleavage of the nascent polypeptide occurred to products which corresponded well with those synthesized m vitro. Significantly, correct processing appeared to be dependent upon a virus-specific product sjmthesized during the translation reaction. 

In summary, the complete translation of EMC virus RNA and correct processing of the polypeptide products appear to occur in mRNA-dependent reticulocyte lysates supplemented with tRNA. Additional data presented at this meeting show that the RNA of another picomavirus, foot-and-mouth disease viams (PAMD), is also completely translated in the same reticulocyte system with excellent fidelity of both translation and processing (L. Black and D. Sangar,... 

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