Chimeric coxsackie B3 virus genomes that express hybrid coxsackievirus-poliovirus 2B proteins: functional dissection of structural domains involved in RNA replication.

The 2B proteins of coxsackievirus and poliovirus (PV) share significant structural similarity and exhibit similar biochemical activities, namely inhibition of protein secretion and modification of membrane permeability. Both proteins contain two hydrophobic domains in the carboxy-terminal two-thirds of their sequence, of which one has the potential to form a cationic amphipathic alpha-helix. To gain more insight into the structural requirements of enterovirus protein 2B for its functioning in viral RNA replication, a chimeric cDNA approach was used. Chimeric coxsackie B3 virus (CBV3) genomes were constructed that expressed either the entire PV 2B protein or hybrid proteins in which specific segments of CBV3 2B were substituted by their corresponding PV counterparts. In vitro synthesis and processing of the chimeric polyproteins showed no abnormalities. CBV3 genomes carrying the entire PV 2B gene failed to replicate. A chimeric genome that expressed a hybrid 2B protein consisting of the amino-terminal one-third of PV and the remainder of CBV3 yielded viable viruses. In contrast, a 2B protein consisting of the amino-terminal one-third of CBV3 and the remainder of PV failed to drive replication. These data imply that a sequence-specific interaction with another viral protein is required to drive RNA replication and suggest that the proposed sites of contact reside in the carboxy-terminal two-thirds of 2B. Hybrid genomes in which either the amphipathic alpha-helix or the other hydrophobic domain was replaced failed to replicate. The potential contribution of these domains to the structure and functioning of protein 2B are discussed.

Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication.

The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.

Coxsackie B3 virus protein 2B contains cationic amphipathic helix that is required for viral RNA replication.

Enterovirus protein 2B has been shown to increase plasma membrane permeability. We have identified a conserved putative amphipathic alpha-helix with a narrow hydrophilic face and an arrangement of cationic residues that is typical for the so-called lytic polypeptides. To examine the functional and structural roles of this putative amphipathic alpha-helix, we have constructed nine coxsackie B3 virus mutants by site-directed mutagenesis of an infectious cDNA clone. Six mutants contained substitutions of the charged residues in the hydrophilic face of the alpha-helix. Three mutants contained insertions of leucine residues between the charged residues, causing a disturbance of the amphipathic character of the alpha-helix. The effect of the mutations on virus viability was assayed by transfection of cells with copy RNA transcripts. The effect on positive-strand RNA replication was examined by introduction of the mutations in a subgenomic luciferase replicon and analysis of luciferase accumulation following the transfection of BGM cells with RNA transcripts. It is shown that both the amphipathy of the domain and the presence of cationic residues in the hydrophilic face of the alpha-helix are required for virus growth. Mutations that disturbed either one of these features caused defects in viral RNA synthesis. In vitro translation reactions and the analysis of viral protein synthesis in vivo demonstrated that the mutations did not affect synthesis and processing of the viral polyprotein. These results suggest that a cationic amphipathic alpha-helix is a major determinant for a function of protein 2B, and possibly its precursor 2BC, in viral RNA synthesis. The potential role of the amphipathic alpha-helix in the permeabilization of cellular membranes is discussed.

PCR-based characterization of Yersinia enterocolitica: comparison with biotyping and serotyping.

PCR-based DNA fingerprinting was used to characterize 48 clinical isolates of Yersinia enterocolitica. The samples were examined by random amplified polymorphic DNA (RAPD-PCR) and inter-repeat PCR (IR-PCR). IR-PCR with two enterobacterial repetitive intergenic consensus primers resulted in patterns which were poorly discriminated; 2 of 11 arbitrary primers (RAPD-PCR) provided sufficient discriminatory power. In comparisons with serotyping and biotyping, RAPD-fingerprinting was the most discriminatory technique and may therefore be a valuable epidemiological tool for the study of Y. enterocolitica infections.

Failure to maintain high-dose treatment regimens during long-term use of zidovudine in patients with symptomatic human immunodeficiency virus type 1 infection.

Long-term tolerance of zidovudine treatment was retrospectively analysed in 97 patients with AIDS or AIDS-related complex. After one year of treatment 68% and after two years 87% of the patients had had at least one dose adjustment during their course of therapy. Myelotoxicity was the most common cause (58% of all cases) of dose reductions and therapy interruptions (dose adjustments). At the time of the first dose adjustment 33 patients (34%) were suffering from anaemia (Hb less than 6.0 g/dl), 20 patients (21%) from leukopenia (leukocytes less than 1.5 x 10(9], and 10 patients (10%) from thrombocytopenia (thrombocytes less than 75 x 10(9]. Fifty-six patients (57%) needed one or more blood transfusions during therapy. The median time from the start of therapy to the time of the first dose adjustment was 14 (range: 2-64) weeks in patients who had a first dose adjustment because of anaemia without co-existing leukopenia or thrombocytopenia, and 37 (range: 6-85) weeks in patients who had a first dose adjustment because of leukopenia without co-existing anaemia or thrombocytopenia (p = 0.01). Peripheral blood CD4 positive lymphocyte counts less than or equal to 100/mm3, anaemia, and CDC classification IV-C1 at the start of treatment were associated with a need for an early dose modification or blood transfusion rather than the need for dose modification per se.