Chimeras of Duck and Heron Hepatitis B Viruses Provide Evidence for Functional Interactions between Viral Components of Pregenomic RNA Encapsidation

Hepadnaviruses, also known as hepatitis B viruses, are DNA viruses that replicate via reverse transcription of an RNA pregenome (pgRNA) (9). The pgRNA is transcribed from the hepadnaviral plasmid present in the nucleus, called covalently closed circular DNA (5, 28). The pgRNA can be translated to produce the viral polymerase, P, and the capsid subunit, C (8, 26). Within the cytoplasm, the pgRNA and P are packaged into capsids in a process known as RNA encapsidation (1, 11). Capsids containing pgRNA and P are competent for reverse transcription of the pgRNA (27). Once DNA synthesis is complete, capsids can be secreted from the cell to produce virions. There are several viral components important for pgRNA encapsidation (Fig. ​(Fig.1).1). The C protein is the subunit of the capsid that contains pgRNA. C protein also might contribute to pgRNA encapsidation directly (21). The P protein is required for pgRNA encapsidation, but the reverse transcriptase or DNA-priming activity of P is not required (1, 11, 29, 30). Also, the pgRNA contains cis-acting sequences that contribute to its encapsidation (6, 12, 16). One sequence, called ɛ, is located near the 5′ end of the pgRNA. ɛ forms a phylogenetically conserved secondary structure that is important for its function (16, 23, 24). For the human hepatitis B virus (HBV), ɛ is the only identified sequence on the pgRNA required for encapsidation (16). FIG. 1. DHBV encapsidation requires the viral P and C proteins and several cis-acting sequences on the pgRNA: ɛ, region II, and possibly the intervening sequence between ɛ and region II. P is represented as an oval, C is represented as a pentagon, ... For the avian hepadnaviruses, a second sequence located about 900 nucleotides (nt) downstream of ɛ, called region II, is required for encapsidation (6, 12). Analysis of duck hepatitis B virus (DHBV) region II showed that sequence from nt 420 to 720 contributes to encapsidation, but deletions within a critical sequence of region II from nt 539 to 610 cause a defect in encapsidation that is similar in magnitude to deletion of ɛ (22). DHBV shares 79% nucleotide identity with heron hepatitis B virus (HHBV), and these viruses have similar encapsidation requirements. The HHBV region II sequence has been roughly mapped to be within nt 398 to 831 (22). Our previous studies have indicated that the intervening sequence between ɛ and region II could also play a role in pgRNA encapsidation (22). In that work, substitutions or large deletions of the intervening sequence between ɛ and region II impaired encapsidation. However, smaller deletions within the intervening sequences did not have an effect on encapsidation. There may be functionally redundant sequences within the intervening sequence, or the intervening sequence may be required as a spacer between ɛ and region II for encapsidation. Previous work with DHBV has demonstrated that P interacts with ɛ to facilitate encapsidation of P and the pgRNA (24). It is not known how region II or the intervening sequence between ɛ and region II contributes to encapsidation; however, it is likely that other interactions are important for encapsidation. One approach to identifying functional interactions is through studies of chimeric viruses. Several groups have used chimeric retroviruses to study the requirements for retroviral encapsidation (3, 4, 7, 17, 19, 25). HHBV/DHBV chimeras have been used successfully to elucidate the interactions between cis-acting sequences that are important for the synthesis of plus-strand DNA (20). Furthermore, our previous work demonstrated that replacing DHBV region II with HHBV region II impaired encapsidation, indicating that HHBV region II is incompatible with one or more DHBV encapsidation components (22). In the present study, we used several chimeras of DHBV and HHBV to identify components that need to be compatible for encapsidation. Understanding these compatibility requirements provides insight into the interactions of viral components required for avian hepadnavirus encapsidation.