Deletion mutants of hepatitis B virus (HBV) are often found in chronically HBV-infected patients. It has not been possible to study the significance of such deletion mutants on liver diseases in a suitable animal model. In this study, we characterized naturally occurring deletion mutants of woodchuck hepatitis virus (WHV) in 11 chronically WHV-infected woodchucks. Deletions within the WHV preS region (nt 2992-338) had a length of 72 or 84 bp and were located in the amino terminal part of preS1. Internal deletions within the core gene (CID) had variable lengths (103 to 312 bp) and were identified within the center of this gene (nt 2021-2587). Four of seven CIDs were in-frame deletions, whereas the remaining three CIDs were out-of-frame deletions and led to the interruption of the reading frame. Sequence analysis of cloned PCR products of CIDs showed that heterogeneous WHV deletion mutants coexisted in single woodchucks. In addition, WHV genomes with double deletions in the preS1 and the core region could be found. We were unable to detect the expression of truncated core proteins in transfection experiments. The CID mutations led to a marked increase of the expression of the luciferase gene which was fused to the start codon of WHV polymerase, probably due to the shortening of the untranslated region or the removal of AUGs preceding the polymerase start codon. The characterization of naturally occurring WHV deletion mutants will allow us to study their biological and pathogenic properties in the woodchuck model in the future.
The diabetogenic variant PV2 of encephalomyocarditis virus was cloned, and three recombinants differing in their 5 poly(C) tracts were analyzed. It is shown that the poly(C) region is not essential for infectivity in mice but does influence the virus load and degree of pathological lesions within the Langerhans' islets but not in the myocardium.Variant PV2 of encephalomyocarditis virus (EMCV) induces an insulin-dependent diabetes mellitus-like syndrome in certain inbred mouse strains by destruction of insulin-producing pancreatic beta cells (14,17). To analyze further the molecular basis of the pathogenic properties, PV2 was cloned and sequenced (17).A feature of murine cardioviruses (EMCV and mengovirus) and bovine aphthoviruses (FMDV) is the homopolymeric cytosine tract (60 to 350 C's) within the 5Ј noncoding region. The length of this region and the distance from the 5Ј end of the RNA depends on virus strain and isolate (3, 4). The task of the poly(C) region in the viral replication cycle is not yet clear; however, it is not likely that it plays a direct role in viral RNA replication or protein synthesis, since cardioviruses with shortened or deleted poly(C) tracts replicate efficiently in cell culture (2,7,10,15). On the other hand, at least in the case of mengovirus the length of the poly(C) tract seems to influence viral pathogenesis upon inoculation into mice, i.e., the fewer the C's, the more attenuated appears the corresponding virus (6,15,18). In contrast to mengoviruses, a full-length poly(C) region seems not to be a prerequisite for infectivity of EMCV applied intracerebrally (10). To further elucidate the function of the poly(C) region for the EMCV life cycle, construction of cDNA clones with modified poly(C) tracts was intended. Since long dC ⅐ dG tracts are neither strictly reproduced from template-dependent polymerases nor precisely maintained in the bacterial host, several approaches have been used to overcome this problem (1,7,16,19,22). To begin with, we constructed and sequenced a full-length cDNA clone of PV2 from overlapping fragments (17). Although the original poly(C) region of EMCV PV2 is composed of 117 or 118 cytosines, after reverse transcription and further cloning procedures the longest poly(C) tract amounted to only 20 cytosines (rPV2/C20 in Fig. 1A). To elongate the 5Ј noncoding region, the EcoRV-HindIII fragment of the clone rPV2/C20 was replaced by the corresponding fragments of pSPORT/EMC-ran or pSORT-EMC-dT, leading to rPV2/ran and rPV2/dT, respectively ( Fig.
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