Genotype-specific complementation of hepatitis delta virus RNA replication by hepatitis delta antigen

Abstract

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.

FIG. 1

Schematic diagram of plasmid constructs used. Thick bars indicate HDV sequences; thin lines represent plasmid sequences. The open segments within the thick bars indicate sequences encoding HDAg. Dashed lines indicate HDV sequences which have been deleted. Numbers indicate sequence coordinates according Wang et al. (32) for genotype I and Casey et al. (7) for genotype III. Rightward-pointing arrows represent cytomegalovirus (CMV) immediate-early promoter. Downward-pointing filled arrowheads and upward-pointing open arrowheads indicate the positions of the antigenomic and genomic RNA autocatalytic cleavage sites, respectively. Ag(−) refers to HDAg-deficient RNAs; otherwise, (−) refers to genomic HDV RNA and (+) refers to antigenomic HDV RNA. Sequences shown directly beneath diagrams of pHDV·I(+)Ag(−) and pHDV·III(−)Ag(−) are for the first six and seven codons, respectively, of the genotype I and III HDAg-coding regions and indicate the position where a mutation was made by inserting a T. Plasmids pHDAg-L·I and pHDAg-L·III, described in Materials and Methods, are the same as their respective counterparts pHDAg-S·I and pHDAg-S·III except that they contain a single nucleotide change which alters the stop codon of HDAg-S from UAG to UGG (Trp) (21). Plasmid pHDV·I(+)ΔApa was described previously as pCMV2-DC-Δ1×1(A) (8). (A) Expression constructs for wild-type genotype III HDV; (B) expression constructs for genotype I and III RNA defective for HDAg; (C) expression constructs for nonreplicating genotype I and III antigenomic RNAs. (D) expression constructs for HDAg.

FIG. 2

RNA replication and HDAg production following transfection with clones of genotype I and genotype III HDV. Huh-7 cells were transfected with pCMV3-DC1×1.2 (left) or pHDV·III(+) (right), which express 1.2 unit-length genotype I and genotype III antigenomic RNA, respectively. Cells were harvested and RNA and proteins were isolated on days 6 (lanes 1 and 4), 9 (lanes 2 and 5), and 12 (lanes 3 and 6). (A) Northern blot analysis of genomic HDV RNA; (B) immunoblot analysis; (C) second run of samples from lanes 3 and 6 of panel B in adjacent lanes in the same gel.

FIG. 3

Genotype-specific support of RNA replication by HDAg-S of genotypes I and III. Huh-7 cells were transfected with indicated constructs for the expression of genomic (left) or antigenomic (right) HDV RNA defective for HDAg production. Cells were also cotransfected with the plasmid vector pCMV3 (lanes −) or plasmids pHDAg-S·I (lanes I) and pHDAg-S·III (lanes III), which express genotype I and III HDAg, respectively. RNAs were isolated 6 days posttransfection and analyzed by Northern blotting. Hybridization probes detected antigenomic RNA (left) or genomic RNA (right).

FIG. 4

Genotype-specific support of RNA replication is exhibited by HDAg clones from multiple genotype I and genotype III isolates. Huh-7 cells were transfected with plasmids pHDV·I(+)Ag(−) (top) and pHDV·III(+)Ag(−) (bottom), which express antigenomic HDV RNA defective for HDAg production. Cells were also cotransfected with expression constructs for HDAg clones from different isolates (lanes 1 to 4, genotype I; lanes 5 to 9, genotype III) or with the plasmid vector pCMV3 (lanes 10). Cotransfected clones: lanes 1, genotype I prototype clone pHDAg-S·I; lanes 2, isolate U02 (7); lanes 3, isolate I27 (26); lanes 4, isolate I43; lanes 5, genotype III prototype clone pHDAg-S·III; lanes 6, isolate Peru 5 (10); lanes 7, isolate Peru 13 (10); lanes 8, isolate P20; lanes 9, isolate P21. RNAs were isolated 7 days posttransfection and analyzed by Northern blotting for HDV genomic RNA. Numbers beneath lanes indicate RNA levels quantified by radioanalytic imaging and normalized for variations in transfection efficiency by evaluating expression of a cotransfected reporter gene (14). Values shown are relative to the prototype (lane 1 in the upper panel; lane 5 in the lower panel), which is assigned a value of 100, and are the averages of two transfections.

FIG. 5

Stabilization of HDV RNA by HDAg. Huh-7 cells were transfected with the indicated expression constructs for HDV antigenomic RNA. Cells were also cotransfected with the plasmid vector pCMV3 (lanes −) or plasmids pHDAg-S·I (lanes I) and pHDAg-S·III (lanes III), which express genotype I and III HDAg, respectively. RNAs were isolated 6 days posttransfection and analyzed for HDV antigenomic RNA by Northern blotting; the hybridization probes did not include HDAg-encoding sequences transcribed by the HDAg expression constructs.

FIG. 6

Support of RNA replication by chimeric genotype I-genotype III HDAg expression constructs. (A) Comparison of HDAg-S sequences of genotype I and III prototype isolates. Uppercase letters for consensus indicate agreement between genotype I and III clones. Dashes in the consensus indicate positions not in agreement; for each isolate, the identities of these positions are indicated in lowercase. Dots indicate gaps introduced to produce an optimal alignment. Lines above sequences indicate boundaries used in the creation of the chimeric constructs (B). Functional domains identified in other studies are indicated in open boxes beneath sequences. The black boxes within the RNA-binding domain indicate arginine-rich motifs (19). (B) Left, schematic representation of HDAg chimeric constructs. Open boxes indicate genotype I sequences, shaded boxes indicate genotype III sequences. Right, relative levels of RNA replication supported by chimeric HDAg constructs. Huh-7 cells were transfected with pHDV·I(+)Ag(−) (genotype I RNA) or pHDV·III(+)Ag(−) (genotype III RNA) and with the indicated HDAg expression constructs. RNAs were isolated 7 days posttransfection and analyzed by Northern blotting for the appropriate genomic HDV RNA. RNA levels were quantified by radioanalytic imaging and normalized for expression of a cotransfected reporter gene (14). Values indicated are relative to the levels of expression obtained with the homologous prototype HDAg clone, as described for Fig. 4. Quantitations presented are averages from two independent transfections and did not vary by more than 10% in separate experiments. Relative activities that were less than 1% of the prototypes are shown as 0.

FIG. 7

Inhibition of genotype I RNA replication by genotype III HDAg. Huh-7 cells were transfected with pCMV3-DC1×1.2 (left) or pHDV·III(+) (right), which express wild-type 1.2 unit-length genotype I and genotype III antigenomic RNA, respectively. Cells were also cotransfected with the indicated HDAg expression constructs or with the plasmid vector pCMV3 (lanes None). RNAs were isolated 6 days posttransfection and analyzed by Northern blotting for HDV antigenomic RNA.