Mengovirus and encephalomyocarditis virus poly(C) tract lengths can affect virus growth in murine cell culture

Many virulent aphthoviruses and cardioviruses have long homopolymeric poly(C) tracts in the 5' untranslated regions of their RNA genomes. A panel of genetically engineered mengo-type cardioviruses has been described which contain a variety of different poly(C) tract lengths. Studies of these viruses have shown the poly(C) tract to be dispensable for growth in HeLa cells, although the relative murine virulence of the viruses correlates directly and positively with tract length. Compared with wild-type mengovirus strain M, mutants with shortened poly(C) tracts grow poorly in mice and protectively immunize rather than kill recipient animals. In the present study, several murine cell populations were tested to determine whether, unlike HeLa cells, they allowed a differential amplification of viruses with long or short poly(C) tracts. Replication and cytopathic studies with four hematopoietically derived cell lines (CH2B, RAW 264.7, A20.J, and P815) and two murine fibroblast cell lines [L929 and L(Y)] demonstrated that several of these cell types indeed allowed differential virus replication as a function of viral poly(C) tract length. Among the most discerning of these cells, RAW 264.7 macrophages supported vigorous lytic growth of a long-tract virus, vMwt (C(44)UC(10)), but supported only substantially diminished and virtually nonlytic growth of vMC(24) (C(13)UC(10)) and vMC(0) short-tract viruses. The viral growth differences evident in all cell lines were apparent early and continuously during every cycle of virus amplification. The data suggest that poly(C) tract-dependent attenuation of mengovirus may be due in part to a viral replication defect manifest in similar hematopoietic-type cells shortly after murine infection. The characterized cultures should provide excellent tools for molecular study of poly(C) tract-mediated virulence.     

Mutational analysis of the mengovirus poly(C) tract and surrounding heteropolymeric sequences.

Previously, we described three mengovirus mutants derived from cDNA plasmids, containing shortened poly(C) tracts (C8, C12, and C13UC10), that exhibited strong attenuation for virulence in mice yet grew like wild-type virus in HeLa cells. Thirteen additional mutants hav now been constructed and characterized. Five of these differ only in poly(C) length, including one with a precise deletion of the tract. The other mutants bear deletions into the regions juxtaposing poly(C). Studies with HeLa cells confirm the essential dispensability of mengovirus's poly(C) tract but reveal a subtle, measurable correlation between poly(C) length and plaque diameter. Virulence studies with mice also revealed a strong correlation between poly(C) length and virulence. For the poly(C)-flanking mutations, the 15 bases directly 5' of the tract proved dispensable for virus viability, whereas the 20 to 30 bases 3' of poly(C) were critical for growth, thus implicating this region in the basal replication of the virus.     

Sequence and location of the poly C tract in aphtho- and cardiovirus RNA.

The poly C tract in the RNA of the aphtho- and cardio viruses has been examined in several isolates of foot-and-mouth disease virus (FMDV) and encephalomyocarditis (EMC) virus. The length of the tract is variable, containing 100 to 170 bases in the FMDV isolates and 80 to 250 bases in the EMC virus isolates. Each poly C tract contains c. 10% A and U residues, located at the 5' end, i.e. most of the tract is a continuous run of C residues. The position of the tract on the genome was the same in each of the FMDV isolates, about 400 bases from the 5' end, whereas in the EMC virus isolates it was about 150 bases from the 5' end.     

Tandem mengovirus 5' pseudoknots are linked to viral RNA synthesis, not poly(C)-mediated virulence.

The RNA genomes from the cardioviruses, hepatoviruses, and aphthoviruses encode two to five tandem pseudoknots within their 5' untranslated regions. These pseudoknots lie adjacent to a pyrimidine-rich sequence, which in cardio- and aphthoviruses takes the form of a homopolymeric poly(C) tract. Seven deletion mutations within mengovirus pseudoknots PK(B) and PK(C) were created and characterized. tested in tissue culture, mengovirus genomes with alterations in PK(C) were viable but had small plaque phenotypes. Larger plaque revertants were isolated and partially characterized, and each proved to be a second-site pseudorevertant with (unmapped) changes elsewhere in the genome. The infectious PK(C) mutant viruses were highly lethal to mice, and deletions in this motif did not affect mengovirus virulence in the same manner as deletions in the adjacent poly(C) tract. In contrast, deletions in PK(B), or deletions which spanned PK(B) + PK(C), produced nonviable genomes. Cell-free translations directed by any of the altered PK sequences gave normal polyprotein amounts relative to wild-type mengovirus. But viral RNA accumulation during HeLa cell infection was dramatically impaired, even with the least disruptive of the PK(C) changes, suggesting the pseudoknots play an essential though undefined role in RNA synthesis and moreover that an intact PK(B) structure is critical to this function.     

3' nontranslated RNA signals required for replication of hepatitis C virus RNA

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.     

Encephalomyocarditis viruses with short poly(C) tracts are more virulent than their mengovirus counterparts.

We have constructed three cDNA clones of encephalomyocarditis virus strain R (EMCV-R) with poly(C) tracts of C4, C9, and C20. RNA transcribed from these cDNAs was infectious to HeLa cells, and the resultant viruses grew well in this system, albeit with plaque sizes that were proportional to the poly(C) length. When injected into mice, the progeny viruses were only slightly less pathogenic than EMCV-R, and the observed degree of attenuation was not nearly as dramatic as for equivalent mengoviruses with similar short poly(C)s. Short-tract poly(C)-mediated attenuation is therefore highly dependent on viral genomic context.     

A wild-type porcine encephalomyocarditis virus containing a short poly(C) tract is pathogenic to mice,pigs, and cynomolgus macaques

Previous studies using wild-type Encephalomyocarditis virus (EMCV) and Mengo virus, which have long poly(C) tracts (61 to 146 C's) at the 5' nontranslated region of the genome, and variants of these viruses genetically engineered to truncate or substitute the poly(C) tracts have produced conflicting data on the role of the poly(C) tract in the virulence of these viruses. Analysis of the nucleotide sequence of an EMCV strain isolated from an aborted swine fetus (EMCV 30/87) revealed that the virus had a poly(C) tract that was 7- to 10-fold shorter than the poly(C) tracts of other EMCV strains and 4-fold shorter than that of Mengo virus. Subsequently, we investigated the virulence and pathogenesis of this naturally occurring short-poly(C)-tract-containing virus in rodents, pigs, and nonhuman primates. Infection of C57BL/6 mice, pigs, and cynomolgus macaques resulted in similar EMCV 30/87 pathogenesis, with the heart and brain as the primary sites of infections in all three animals, but with different disease phenotypes. Sixteen percent of EMCV 30/87-infected pigs developed acute fatal cardiac failure, whereas the rest of the pigs were overtly asymptomatic for as long as 90 days postinfection (p.i.), despite extensive myocardial and central nervous system (CNS) pathological changes. In contrast, mice infected with >/==" BORDER="0">4 PFU of EMCV 30/87 developed acute encephalitis that resulted in the death of all animals (n = 25) between days 2 and 7 p.i. EMCV 30/87-infected macaques remained overtly asymptomatic for 45 days, despite extensive myocardial and CNS pathological changes and viral persistence in more than 50% of the animals. The short poly(C) tract in EMCV 30/87 (CUC(5)UC(8)) was comparable to that of strain 2887A/91 (C(10)UCUC(3)UC(10)), another recent porcine isolate.     

Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA

An mRNA-dependent reticulocyte lysate has been used to translate foot-and-mouth disease virus RNA in vitro. Polypeptides P16, P20a, and P88, which have been shown to be derived from the 5' end of the RNA by pactamycin mapping experiments with infected cells, were preferentially synthesized in vitro. Removal of VPg, the small protein covalently linked to the 5' end of the genome RNA, had no effect on the translation of the RNA. The two RNA fragments (L and S) produced by specific digestion of the polycytidylic acid [poly(C)] tract with RNase H were also translated in vitro. The L fragment, consisting of RNA to the 3' side of the poly(C) tract and including the polyadenylic acid [poly(A)] tract, directed the synthesis of the same products as those made by full-length RNA. However, no small defined products were produced when the S fragment, which contains the 5' end of the RNA, was translated. These results show that the major initiation site for protein synthesis on foot-and-mouth disease virus RNA is to the 3' side of the poly(C) tract. Furthermore, the use of N-formyl [35S]methionine tRNAfMet as a label for the initiation peptides showed that the major polypeptide labeled in lysates primed with both full-length RNA and the L fragment was P16, i.e., the protein nearest the initiation site for translation as deduced from pactamycin mapping experiments. Fragments of RNA were also translated in vitro. Those containing the poly(C) tract gave products similar to those produced when full-length RNA was translated. The polypeptides synthesized when fragments containing the poly(A) tract were used, however, did not resemble those made from full-length RNA.     

Replication of Mengovirus in HeLa Cells Preinfected with Nonreplicating Poliovirus

The replication of mengovirus in HeLa cells preinfected with poliovirus in the presence of 10(-3) M guanidine was investigated. Although host cell protein synthesis is inhibited by the presence of nonreplicating poliovirus, it is found that mengovirus ribonucleic acid (RNA) and protein synthesis proceed normally under the same conditions. Furthermore, no effects on mengovirus growth by poliovirus can be detected either when Mengo protein synthesis is interrupted by Acti-Dione or when its RNA synthesis is reduced by incubation at 28 C. It is suggested that the poliovirus inhibitory factor may be able to distinguish between an RNA element required in the protein-synthesizing apparatus of the host cell and a comparable element in that of the heterologous virus.     

Nucleotide sequences in the RNA of mammalian leukemia and sarcoma viruses.

The nucleotide sequences in viral RNA from purified murine sarcoma and hamster leukemia viruses (S+H+) from HTG-1 cells and Rauscher leukemia virus (RLV) from JLS-V 9 cells have been examined by polynucleotide agarose affinity chromatography. There is at least one copy of poly(A) sequences per genomic viral RNA molecule. After heat denaturation of genomic viral RNA (S+H+), there are two types of viral subunits for 34S and 28S species: one that contains poly(A) sequences and one that does not. There are no detectable poly(U) tracts in the viral RNA. However, poly(C) sequences and poly(G) tracts were detected in viral RNA, although less poly(G) than poly(C) tracts were observed. In addition, heat-denatured genomic viral RNA has a greater affinity for poly(G) agarose column than native genomic viral RNA.     

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.     

The nucleotide sequence at the 5'' end of foot and mouth disease virus RNA.

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.     

An investigation of the stability of messenger RNAs in cell-free,translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells

The stabilities and translation of Ehrlich ascites tumor cell poly(A)-containing mRNA and mengovirus RNA in fractionated cell-free protein synthesizing systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells were studied. During incubation of the systems about 20% of the input RNA is reduced in size and associated with ribosomes engaged in polypeptide synthesis; the remainder is rapidly degraded by RNases. At the end of active translation, both mRNA and nascent proteins are bound to polysomes which are of the same size as those formed during active protein synthesis. The kinetics of protein synthesis closely follow those of RNA hydrolysis. The stabilities of mengovirus RNA and poly(A)-containing mRNA from Ehrlich ascites tumor cells are the same in both systems.     

Shutoff of HeLa cell protein synthesis by encephalomyocarditis virus and poliovirus: a comparative study.

Previous experimental results have suggested that poliovirus and encephalomyocarditis (EMC) virus employ very different mechanisms for shutting off host protein synthesis. However, this conclusion is suspect, inasmuch as different cell types were used for the two viruses; hence the apparent mechanistic differences might be specific for cell type and not virus type. To test this possibility we compared shutoff mechanisms in poliovirus- and EMC virus-infected HeLa cells. Striking differences were seen: poliovirus-induced shutoff was much more rapid and extensive than that induced by EMC virus; relative translation rates of certain host proteins were inhibited to different extents by the two viruses; initiation factors prepared from poliovirus-infected cells were specifically defective for translation of capped mRNA's in vitro, whereas those from EMC virus-infected cells were not. These results indicate that EMC virus and poliovirus employ different mechanisms for the shutoff of HeLa cell protein synthesis. This conclusion is consistent with much earlier work and indicates that many differences previously reported are specific to virus type.     

Induction of 2',5' oligo(A) synthetase in tumor-bearing mice with encephalomyocarditis (EMC) virus or poly(I)poly(C)

Infection of 13 month-old C3H mice with EMC virus or inoculation with the interferon inducer poly(I)poly(C) results in elevated levels of the enzyme 2',5' oligo(A) synthetase only in animals with spontaneous tumors (breast cancer or hepatomas). High enzymatic activities are detected in homogenates from liver, spleen, plasma and neoplastic cells of the animals with breast carcinomas and only in the neoplastic liver cells of the animals with hepatomas.     

Construction and characterization of two infectious molecular clones of encephalomyocarditis virus.

We constructed and characterized two infectious molecular clones of encephalomyocarditis (EMC) virus. Both constructs, pDL and pDA, were assembled from five overlapping cDNA clones derived from the diabetogenic variant of EMC virus (EMC-D) and from two synthetic oligonucleotide cartridges. pDA contained a single point mutation at position 1720 within the "puff" region of capsid protein 1AB that was derived from the nondiabetogenic variant of EMC virus (EMC-B). This point mutation resulted in an amino acid substitution of arginine (EMC-B) for lysine (EMC-D). Our construction illustrates two novel findings: (i) that the problem of stably cloning long poly(C) tracts of EMC virus can be circumvented by the use of a shortened, synthetic, poly(dC-dG) oligonucleotide cartridge, and (ii) that a single point mutation in the puff region of the capsid protein 1AB leads to change in its electrophoretic mobility and to a change in the plaque size of recombinant virus.