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Cell lines persistently infected with foot-and-mouth disease virus (FMDV) have been established by growth of BHK-21 (c-13) or IBRS-2 (c-26) that survived standard cytolytic infections with FMDV. They maintain cytoplasmic FMDV RNA sequences, as shown by dot blot hybridization tests, using cloned FMDV eDNA as probes. Cell line C1-BHK-Rc1 was derived by infection of cloned BHK-21 c1 cells and plaque-purified FMDV C-S8 c1. Indirect immunofiuorescence assays indicated the presence of FMDV antigens. It was resistant to superinfection by FMDV C-S8 c1, O-S7, or A5, but not by encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or Semliki forest virus (SFV). Infectious FMDV was detected in the culture medium only up to cell passage 65. The virus isolated from C1-BHK-Rc1 cells showed decreased plaque size and diminished yield in infections at 42°. Multiple mutations in the intracellular FMDV RNA have been detected by T1 oligonucleotide fingerprinting of genomic RNA segments hybridized to FMDV cDNA fragments. At late cell passages, when no infectious FMDV is detected, cells continue to express viral antigens and FMDV RNAs with deletions of up to 3 kb have been identified by Northern blot analysis. We conclude that persistent infections of cell cultures with FMDV are readily established and that multiple genetic and phenotypic variations occur in the virus during persistence.

The complete genetic information for the neuraminidase (NA) gene of influenza virus A/Bangkok/1/79 has been cloned by in vitro synthesis of dsDNA, insertion into pBR322 plasmid, and transformation of Escherichia coli The nucleotide sequence of the NA gene has been determined by the Maxam and Gilbert method. It is 1466 nucleotides long and contains a single open reading frame with a coding capacity for 469 amino acids. When compared to the NA genes of the N2 strains A/Victoria/3/75, A/Udorn/72, A/NT/60/ 68, and A/RI/5 − /57, 90% of the nucleotide positions and 87% of the amino acid positions remained invariant. Forty-two nucleotide changes and 14 amino acid changes accumulated in the period 1975–1979, but the general structure of the protein appeared to remain constant.

When influenza virus monoclonal antibody-resistant (mar) mutants are selected by incubation in vitro with excess antibody, 90 to 99% of the mutants are not detectable. This observation may be explained by encapsidation of mar mutant RNAs within phenotypically wild-type envelopes. This phenotypic hiding can be revealed by selection of mar mutants in vivo after virus uncoating. Using experimental procedures appropriate to detect all viable mar mutants in a virus population, we determined precisely the mutation rates to the mar genotype by the fluctuation test for two nonoverlapping monoclonal antibodies.

The DNA of the B. subtilis phage ø29 has been described as unpermuted linear duplex molecules1 of molecular weight 11 '— 106, but the formation of circular molecules has also been indicated, suggesting the existence of cohesive ends1,2., This investigation was supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research (to E. V. and M. S.) and from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina (to C. V.). J. O. is a fellow ofFondo Nacional para la Formación de Personal Investigador. Sorne of the electron micrographs were made in the Department of Biochemistry, New York University School of Medicine. We thank Dr A. K. Kleinschmidt for the facilities given and for critical reading of the manuscript.

5 pages, 5 figures.-- PMID: 8995663 [PubMed].-- PMCID: PMC191194.-- Full-text version available Open Access at the journal site., The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ- L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA., B.P. was a fellow from Programa Nacional de Formación de Personal Investigador. This work was supported by Programa Sectorial de Promoción General del Conocimiento (grant PB94-1542), Comunidad Autónoma de Madrid (grant A0063), and the EU HCM Program (grant ERBCHRXCT949453).

Temperature-sensitive COS cells, clone E540, have been stably transformed at a restrictive temperature with plasmid pVA1, which contains the adenovirus type 5 virus-associated (VA) genes in addition to the Neor marker. Transformed cell clones, named EVA cells, contained adenovirus DNA in an integrated form while grown at restrictive temperature but accumulated up to 100 to 200 copies of the input plasmid per cell after temperature shift down. Concomitant with this gene amplification, an accumulation of VA RNA was observed, reaching average concentrations of 10(4) to 10(5) copies per cell. The VA RNA synthesized in EVA cells is functional, as judged by inhibition of in vitro eucaryotic initiation factor-2 phosphorylation and enhancement of reporter gene expression. These EVA cell lines may be of use to study the mechanism of VA RNA function in the absence of adenovirus infection.

The establishment and maintenance of asymmetries in certain cells implies the localized expression of many proteins, a property often enhanced by the localization of the corresponding mRNAs (for reviews, see references 2 and 47). The relevance of mRNA localization at precise sites of the cell in the definition of polarity of developing embryos has been documented in both Drosophila melanogaster and Xenopus laevis. Thus, the positions of gurken and oskar mRNAs in the fly oocyte define its dorsoventral axis (36) and the location of the pole plasm at the posterior pole (15), respectively. Likewise, the localization of the bicoid and nanos mRNAs at the anterior and posterior poles of the embryo, respectively, leads to the generation of two opposing gradients of their protein products and ultimately to the definition of the head, thorax, and abdomen of the embryo (50). In the case of X. laevis, several mRNAs, such as Xcat2, Xcat3, and Xlsirt, are directed to the germ plasm (34), while others, such as Vg1 and Xwnt11, accumulate at the vegetal cortex (30). By analogy to the D. melanogaster genes, these mRNAs are thought to play a role in defining patterns in the X. laevis embryo. In fact, a region of the Xcat2 protein shows sequence homology to the zinc finger domain of nanos protein (34). The specific localization of certain mRNAs to precise sites within the cell is not an exclusive property of germ cells or developing embryos. A number of observations indicate that some of the mRNAs of somatic cells are also localized at different sites within the cell. Thus, myelin-binding protein is translated in oligodendrocytes from free ribosomes on localized mRNA (51), and the microtubule-associated protein MAP2 is translated preferentially in dendrites of the neurons (23). Likewise, different actin protein isoforms are translated from their mRNAs at distinct sites of myoblasts (27). In some cases, the intracellular localization of mRNAs involves cis-acting signals consisting of distinct secondary structures of their untranslated regions (UTRs) (reviewed in reference 47). These cis signals are thought to mediate localization by interaction with RNA-binding proteins, the best studied of which is the Staufen protein of D. melanogaster (dmStaufen) (48; reviewed in reference 50). dmStaufen protein is the product of a maternal mRNA of D. melanogaster that is involved in the accumulation of bicoid and oskar mRNAs at the anterior pole of the embryo and the posterior pole of the oocyte, respectively (48). It contains double-stranded RNA (dsRNA)-binding domains that associate with bicoid mRNA through a precise secondary structure in its 3′ UTR (18). Our group has been studying the influenza virus nonstructural protein NS1, an RNA-binding protein (26, 33, 42) that may be involved in several regulatory processes during viral infection (reviewed in reference 37). These include the modulation of pre-mRNA splicing (20, 21, 32), the retention of poly(A)-containing RNA in the nucleus (20, 42), and the stimulation of viral mRNA translation (10, 14). These properties of NS1 protein seem related to particular interactions with certain cellular or viral RNA molecules and may also involve interaction with specific cellular factors (26, 40, 42, 43). In the course of a genetic screen to detect candidate cellular proteins that may pertain to these biochemical effects, we identified a human gene with homology to the dmStaufen gene. The encoded protein (hStaufen-like) was localized by immunofluorescence to the rough endoplasmic reticulum in cultured cells, was associated with polysomes, and behaved like an RNA-binding protein.

The effect of NS1 protein on the efficiency of influenza virus mRNA translation was evaluated by determining the accumulation of nucleoprotein (NP) or M1 mRNAs in the cytoplasm of cells expressing either of these genes alone or in combination with the NS1 gene, as well as the total cell accumulation of NP or M1 protein. Coexpression of NS1, but not of NS2 protein, led to increases in the translation of these mRNAs in the range of 5- to 100-fold. This translation enhancement was specific for viral mRNAs, since the translation of neither cat nor lacZ mRNAs was affected by the coexpression of NS1 protein. The use of chimeric cat genes containing the 5'-extracistronic sequences of the influenza virus mRNAs corresponding to segment 2, 7, or 8 indicated that these sequences can in part account for the observed effect. The enhancement of viral mRNA translation mediated by NS1 protein was due to an increase in the translation initiation rate, since the sizes of NP-specific polysomes, but not those of lacZ-specific polysomes, was significantly higher in cells coexpressing NS1 protein than in those expressing only the NP gene.

The genetic heterogeneity generated upon passage of foot-and-mouth disease virus (FMDV) in cell culture has been evaluated by T1-oligonucleotide fingerprinting of genomic RNA. Plaque-purified FMDV O-S7 and C-S8 were propagated by serial low multiplicity infections of BHK-21 (c-13) or IBRS-2 (c-26) cells. In independent parallel passage of the same virus, different oligonucleotide variations were fixed in the RNAS. T1-oligonucleotide fingerprinting of RNA from 34 individual viral clones derived from two passaged populations shows an extensive heterogeneity, with some mutations present in only one of the cloned genomes analyzed. Some FMDV variants are phenotypically distinct in that they yield increased progeny in infections of cell monolayers. From the number of variant sequences it can be estimated that each infectious RNA in the population differs in two to eight mutations from the average parental sequence. Thus, passaged FMDV populations consist of a pool of variants, an observation previously made with phage Qβ ( E. Domingo, D. Sabo, T. Taniguchi, and C. Weissmann, Cell 13 , 735–744, 1978 ). The FMDV genome must be described. as a fluctuating distribution of sequences due to its high mutability. This may be the basis of the extensive genetic and antigenic diversity of this virus in nature.

The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.

The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype. Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.