Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation.

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.     

Characterization of a pre-S polypeptide on the surfaces of infectious avian hepadnavirus particles.

DNA from the pre-S region of the duck hepatitis B virus (DHBV) genome was inserted into an open reading frame vector designed to give high-level expression in Escherichia coli. The resulting fusion protein contained the first 8 amino acids of beta-galactosidase, 86 amino acids of the DHBV pre-S region, and 219 amino acids of chloramphenicol acetyltransferase at the C terminus (beta-gal:pre-S:CAT). Rabbit antiserum against purified beta-gal:pre-S:CAT was used to identify pre-S-containing polypeptides in DHBV particles by Western blotting. A dominant species of 36 kilodaltons (kDa) was identified. Antiserum against the major 17-kDa DHBsAg polypeptide also reacted with the 36-kDa protein. This suggests that the DHBV envelope gene polypeptides share the same carboxyl terminus, but differ in the sites from which translation is initiated. N-linked carbohydrate was not detected on either the 17- or 36-kDa envelope proteins. Anti-beta-gal:pre-S:CAT abolished infectivity of the virus in an in vitro assay. Thus, the pre-S region is exposed on the surfaces of infectious virions and may be directly involved in binding of virus to host-cell receptors.     

Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2.

We used a series of deletion mutations in the untranslated leader region of human immunodeficiency virus type 2 (HIV-2) to seek cis-acting packaging signals. Sequences between the 5' major splice donor and the gag initiation codon, where such signals have been identified in HIV-1, appear to make a measurable but very minor contribution to genomic RNA packaging, and deletions here had little effect on viral replication in vitro. Immediately 5' to the splice donor, two regions were identified which, when deleted, caused a significant replication defect. The most proximal of these to the splice donor demonstrated a phenotype consistent with its being a major cis-acting packaging signal in HIV-2.     

A cis-acting DNA signal for encapsidation of simian virus 40.

Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protein-protein interactions in the formation of a unique three-dimensional structure around the viral minichromosome. A pseudoviral system developed in our laboratory, in which the viral early and late gene products are supplied in trans (by helpers), was used to analyze the encapsidation process independent of viral gene expression. With this experimental system we have discovered a requirement for a specific DNA signal for encapsidation, ses (for simian virus 40 encapsidation signal).ses is present within a 200-bp DNA fragment, which includes, in addition to the viral origin of replication (ori), six GGGCGG repeats (GC boxes) and 26 bp of the enhancer element. Deletion of the GC boxes and the enhancer sequences almost abolished encapsidation, while DNA replication was only moderately decreased. The ability to encapsidate was not regained by reinserting a DNA fragment carrying ses in the sesdeleted plasmid 2 kbp away from the ori, suggesting that for encapsidation the two DNA elements have to be close to each other. These findings afford novel strategies for the investigation of viral encapsidation.     

Frequency of spontaneous mutations in an avian hepadnavirus infection

In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 x 10(-5) to 6 x 10(-5) per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 x 10(-5) to 4.5 x 10(-5) per nucleotide per generation.     

A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.     

Recognition of RNA encapsidation signal by the yeast L-A double-stranded RNA virus

The encapsidation signal of the yeast L-A virus contains a 24-nucleotide stem-loop structure with a 5-nucleotide loop and an A bulged at the 5' side of the stem. The Pol part of the Gag-Pol fusion protein is responsible for encapsidation of viral RNA. Opened empty viral particles containing Gag-Pol specifically bind to this encapsidation signal in vitro. We found that binding to empty particles protected the bulged A and the flanking-two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals. The five nucleotides of the loop sequence ((4190)GAUCC(4194)) were not protected. However, T1 RNase protection and in vitro mutagenesis experiments indicated that G(4190) is essential for binding. Although the sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C(4194) was also directly involved in binding to empty particles rather than indirectly through its potential base pairing with G(4190). These results suggest that the Pol domain of Gag-Pol contacts the encapsidation signal at two sites: one, the bulged A, and the other, G and C bases at the opening of the loop. These two sites are conserved in the encapsidation signal of M1, a satellite RNA of the L-A virus.     

Characterization of a murine coronavirus defective interfering RNA internal cis-acting replication signal.

The mouse hepatitis virus (MHV) sequences required for replication of the JHM strain of MHV defective interfering (DI) RNA consist of three discontinuous genomic regions: about 0.47 kb from both terminal sequences and a 0.13-kb internal region present at about 0.9 kb from the 5' end of the DI genome. In this study, we investigated the role of the internal 0.13-kb region in MHV RNA replication. Overall sequences of the 0.13-kb regions from various MHV strains were similar to each other, with nucleotide substitutions in some strains; MHV-A59 was exceptional, with three nucleotide deletions. Computer-based secondary-structure analysis of the 0.13-kb region in the positive strand revealed that most of the MHV strains formed the same or a similar main stem-loop structure, whereas only MHV-A59 formed a smaller main stem-loop structure. The RNA secondary structures in the negative strands were much less uniform among the MHV strains. A series of DI RNAs that contained MHV-JHM-derived 5'- and 3'-terminal sequences plus internal 0.13-kb regions derived from various MHV strains were constructed. Most of these DI RNAs replicated in MHV-infected cells, except that MRP-A59, with a 0.13-kb region derived from MHV-A59, failed to replicate. Interestingly, replication of MRP-A59 was temperature dependent; it occurred at 39.5 degrees C but not at 37 or 35 degrees C, whereas a DI RNA with an MHV-JHM-derived 0.13-kb region replicated at all three temperatures. At 37 degrees C, synthesis of MRP-A59 negative-strand RNA was detected in MHV-infected and MRP-A59 RNA-transfected cells. Another DI RNA with the internal 0.13-kb region deleted also synthesized negative-strand RNA in MHV-infected cells. MRP-A59-transfected cells were shifted from 39.5 to 37 degrees C at 5.5 h postinfection, a time when most MHV negative-strand RNAs have already accumulated; after the shift, MRP-A59 positive-strand RNA synthesis ceased. The minimum sequence required for maintenance of the positive-strand major stem-loop structure and biological function of the MHV-JHM 0.13-kb region was about 57 nucleotides. Function was lost in the 50-nucleotide sequence that formed a positive-strand stem-loop structure identical to that of MHV-A59. These studies suggested that the RNA structure made by the internal sequence was important for positive-strand MHV RNA synthesis.     

Specific encapsidation of fragments of TMV RNA.

The in vitro reconstitution of tobacco mosaic virus (TMV) is initiated by the binding of a disk of TMV protein to the 'disk recognition site', a region of the RNA chain at or near the 5'-terminus for which the disk has special affinity. In order to gain insight into the recognition process, we have studied the ability of disks to encapsidate short RNA fragments produced by partial pancreatic or T1 RNase digestion of TMV RNA. The disk is capable of dicriminating among such fragments, encapsidating only a few of the many present in the digest. The products of encapsidation are short nucleoprotein rods of the same diameter as TMV and of length proportional to that of the encapsidated RNA fragment. The particles differ from TMV, however, in one significant aspect (apart from their length): they possess rings of RNA-free protein at one or both extremities of the rod. In the case of T1 RNase digestion the principal encapsidated fragments were fragments T1 (105 nucleotides) and a family of smaller fragments containing elements of the same sequence. Partial digestion with pancreatic RNase generated only one major fragment (fragment P1; 150 nucleotides) with affinity for the disk. Fragment T1 has been sequenced and shown to represent a portion of the coat protein cistron. Fragment P1 has been partially sequenced but its function is not yet known. Several lines of evidence indicate that fragment T1 is not the disk recognition site. The portion of the TMV RNA chain from which fragment P1 is derived, on the other hand, is encapsidated early in the reconstitution process; thus fragment P1 may contain the disk recognition site. Fragment T1 and fragment P1 both have purine-rich and cytosine-poor sequences near their termini. In addition, fragment T1, and possibly fragment P1, possess a periodicity of order three in purine residues. It seems likely that one or both of the aforesaid properties are largely responsible for the affinity of these fragments for the disk.     

Low dynamic state of viral competition in a chronic avian hepadnavirus infection

The dynamic state of infection of 11 ducks with the duck hepatitis B virus was investigated. Chronic infections were established in newly hatched ducklings by inoculation with a mixture of wild-type virus and a mutant virus with a partial replication defect. As expected, the wild-type virus was rapidly enriched in the virus population during the spread of infection. Enrichment thereafter was correlated with normal growth of the liver, with the average mutant-to-wild-type ratio stabilizing for at least 2 months beyond the time at which the liver mass stabilized. Using experimentally determined growth rates for the mutant and wild-type viruses, we estimated that after the spread of infection, competition between the two virus strains was limited by the amount of replication required to infect new hepatocytes in the growing livers. The results suggest that, in a chronically infected liver, the selection of variants with a replication rate advantage is inefficient and that the emergence of such variants would depend on induced liver cell turnover, such as that occurring during chronic hepatitis.