Over the last few years, an essential RNA structure known as the cis-acting replicative element (cre) has been identified within the protein-coding region of several picornaviruses. The cre, a stem-loop structure containing a conserved AAACA motif, functions as a template for addition of U residues to the protein primer 3B. By surveying the genomes of representatives of several serotypes of foot-and-mouth disease virus (FMDV), we discovered a putative cre in the 5 untranslated region of the genome (contiguous with the internal ribosome entry site [IRES]). To confirm the role of this putative cre in replication, we tested the importance of the AAACA motif and base pairing in the stem in FMDV genome replication. To this end, cre mutations were cloned into an FMDV replicon and into synthetic viral genomes. Analyses of the properties of these replicons and genomes revealed the following. (i) Mutations in the AAACA motif severely reduced replication, and all viruses recovered from genomes containing mutated AAACA sequences had reverted to the wild-type sequence.(ii) Mutations in the stem region showed that the ability to form this base-paired structure was important for replication. Although the cre was contiguous with the IRES, the mutations we created did not significantly reduce IRES-mediated translation in vivo. Finally, the position of the cre at the 5 end of the genome was shown not to be critical for replication, since functional replicons and viruses lacking the 5 cre could be obtained if a wild-type cre was added to the genome following the 3D pol coding region. Taken together, these results support the importance of the cre in replication and demonstrate that the activity of this essential element does not require localization within the polyprotein-encoding region of the genome.Foot-and-mouth disease, one of the most important known pathogens of livestock, is caused by a small RNA virus of the family Picornaviridae. Foot-and-mouth disease virus (FMDV) is the prototype member of the Aphthovirus genus of this family, and although aspects of FMDV replication resemble those of many other picornaviruses, there are notable differences between FMDV and other viruses that include FMDV's broad host range and several unique genetic features.The FMDV genome is over 8,300 bases in length and is covalently bound at its 5Ј terminus to a 23-to 24-amino-acid genome-linked protein, 3B. The genome encodes three copies of 3B, all of which are apparently utilized (8,15). No natural isolates of FMDV with fewer than three 3B coding regions have been identified, suggesting that there is a strong selective pressure maintaining this redundancy, since homologous recombination within the FMDV genome (22) should readily eliminate redundant copies of 3B. Despite the observed retention of three 3Bs in all natural FMDVs, viruses lacking one of the 3Bs or with two nonfunctional 3Bs have been derived by genetic engineering (7). Recently, we have observed that viruses lacking two of the three 3Bs can be generated by similar methodology, an...

In 1997, an epizootic in Taiwan, Province of China, was caused by a type O foot-and-mouth disease virus which infected pigs but not cattle. The virus had an altered 3A protein, which harbored a 10-amino-acid deletion and a series of substitutions. Here we show that this deletion is present in the earliest type O virus examined from the region (from 1970), whereas substitutions surrounding the deletion accumulated over the last 29 years. Analyses of the growth of these viruses in bovine cells suggest that changes in the genome in addition to the deletion, per se, are responsible for the porcinophilic properties of current Asian viruses in this lineage.

The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cattle, pigs, sheep, goats, and many wild animals. The disease is characterized by fever and vesicular lesions of the epithelium of the mouth, tongue, feet, and teats. The causal agent, FMD virus (FMDV), is a positive-stranded RNA virus that is the type species of the Aphthovirus genus of the Picornaviridae. The FMDV genome is over 8 kb in length and contains a protein cap (3B, also known as VPg) (27). During replication, the genome is expressed as a single open reading frame (ORF) that is processed into mature polypeptide products. Translation of the ORF begins with a proteinase (L pro ), which is followed in the ORF by the structural proteins (1A, 1B, 1C, and 1D), a short autoproteinase (2A), and the remaining nonstructural proteins (2B, 2C, 3A, 3B, 3C pro , and 3D pol ). 3C pro is responsible for proteolytic cleavage of the majority of the cleavage sites in the FMDV polyprotein (33), and 3D pol is the core subunit of the picornavirus RNA-dependent RNA polymerase (7). Protein 3B, which is represented in three nonidentical copies in FMDV (8), is covalently bound to the 5Ј end of the genome and antigenome, and functions in priming picornavirus RNA synthesis (see reference 34 for review). The functions of the nonstructural proteins 2B, 2C, and 3A are less well understood, although all three have hydrophobic domains (9, 26), and all have been found physically associated with intracellular membranes that proliferate in picornavirus-infected cells (2,3,29,30,32).We have previously shown that a deletion in the 3A protein of FMDV is a characteristic of a virus (O/TAW/97) that devastated the Taiwanese pork industry in 1997 and that the deleted 3A is responsible for the virus' inability to cause disease in cattle (1). However, this deleted 3A does not interfere with production of an acute and highly and readily transmissible disease in pigs (5). The deletion in O/TAW/97 occurs at positions 93 to 102 of the 153-amino-acid 3A protein and is similar in size and position to deletions that were found in eggadapted derivatives of FMDV that were developed for use as vaccines in South America (12). Interestingly, an investigation of the 3A coding regions of a number of Asian serotype O FMDVs revealed that viruses harboring this deletion have been circulating in Asia for more than 30 years and that viruses with 3As harboring a deletion spanning residues 133 to 143 have been circulating in Southeast Asia since the mid 1990s (18). Recently, Sobrino and coworkers have shown that adaptation of an FMDV isolate to cause disease in guinea pigs is dependent on a point mutation in 3A, adding further supp...

Picornavirus infection induces the proliferation and rearrangement of intracellular membranes in response to the synthesis of nonstructural proteins, including 3A. We have previously shown that changes in 3A are associated with the inability of a Taiwanese strain of foot-and-mouth disease virus (FMDV) (OTai) to grow in bovine cells and cause disease in cattle, although the virus grows to high titers in porcine cells and is highly virulent in pigs (C. W. Beard and P. W. Mason, 2000, J. Virol. 74, 987-991). To study if differences in the distribution of 3A could account for the species specificity of OTai, we compared the localization of the OTai 3A with a bovine-virulent 3A (serotype A12) in keratinocytes prepared from the tongues of cattle and pigs. Following either infection of keratinocytes or transfection with 3A we were unable to discern differences in 3A distribution in either species of keratinocyte, independent of the strain of virus (or 3A) utilized. In both cell types, 3A distributed in a pattern that overlapped with an endoplasmic reticulum (ER) marker protein, calreticulin (CRT). Furthermore, although FMDV infection or transfection with 3A did not result in a gross redistribution of CRT, both virus infection and 3A transfection disrupted the Golgi. Other picornaviruses that disrupt Golgi function are sensitive to brefeldin A (BFA), a fungal metabolite that interferes with retrograde transport between the Golgi and the ER. Interestingly, BFA has little effect on FMDV replication, suggesting that FMDV may acquire cellular membranes into its replication complexes in a manner different from that of other picornaviruses.

Foot-and-mouth disease virus (FMDV) initiates infection by binding to integrin receptors via anFoot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease (FMD), a highly contagious disease of cloven-hoofed animals, including cattle, swine, goats, sheep, and other species of wild ruminants. The virus exhibits a remarkable adaptation and serological diversity exemplified by the existence of a large number of subtypes within each serotype and the fact that infected animals can also become persistent carriers (2,3,27,49). In infected animals the virus replicates very rapidly and spreads among in-contact susceptible animals by aerosol or direct contact. Clinical signs of FMD, including vesicles on the feet and the mouth, can appear as early as 2 days after virus exposure (reference 20 and references therein).FMDV is the type species of the Aphthovirus genus of the family Picornaviridae. The viral genome, consisting of a single open reading frame, is flanked by a long 5Ј-nontranslated region (NTR) linked to a small viral peptide, VPg, and a short 3ЈNTR followed by a poly(A) tail (Fig. 1a). Four structural proteins (VP1 to VP4) constitute the viral capsid, and a number of essential nonstructural proteins, including the RNAdependent RNA polymerase (3D pol ), are involved in the replication of viral RNA (reviewed in references 7 and 45).The first step in FMDV infection involves the recognition of a cell surface receptor. An Arg-Gly-Asp (RGD) amino acid sequence located within the ␤G-␤H (G-H) loop of the capsid protein VP1 (1, 28, 30) is known to bind to several integrins of the ␣ V subgroup, including ␣ V ␤ 1 , ␣ V ␤ 3 , ␣ V ␤ 6 , and ␣ V ␤ 8 , and is involved in virus attachment to cells and interaction with neutralizing antibodies (8, 18, 21, 24-26, 34, 36, 39, 40, 52). The generation of genetically engineered virions carrying deletions or mutations of the RGD motif leads to virus which is unable to bind to cultured cells or cause disease in susceptible animals (29,34,37,43).In cell culture, FMDV binds preferentially to one or more of the above-mentioned integrins with different specificities, and integrin utilization varies between serotypes (7, 17, 18). The reason for this specificity is not clearly understood; however, it is known that sequences surrounding the RGD motif, or located in other regions of the viral capsid, could induce structural changes in the G-H loop of VP1 and modulate binding specificity (14,42). These changes may occur with the replacement of only one or a few amino acids at the surface of the virus (reviewed in reference 7). Viruses propagated in vitro can exploit alternative mechanisms to bind and enter the host cell independent of integrin binding. Among them, the use of heparan sulfate has been correlated with the acquisition of positively charged amino acids on the virus capsid surface (19,

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