Chimeras of duck and heron hepatitis B viruses provide evidence for functional interactions between viral components of pregenomic RNA encapsidation

Abstract

Packaging of hepadnavirus pregenomic RNA (pgRNA) into capsids, or encapsidation, requires several viral components. The viral polymerase (P) and the capsid subunit (C) are necessary for pgRNA encapsidation. Previous studies of duck hepatitis B virus (DHBV) indicated that two cis-acting sequences on pgRNA are required for encapsidation: epsilon, which is near the 5' end of pgRNA, and region II, located near the middle of pgRNA. Later studies suggested that the intervening sequence between these two elements may also make a contribution. It has been demonstrated for DHBV that epsilon interacts with P to facilitate encapsidation, but it is not known how other cis-acting sequences contribute to encapsidation. We analyzed chimeras of DHBV and a related virus, heron hepatitis B virus (HHBV), to gain insight into the interactions between the various viral components during pgRNA encapsidation. We learned that having epsilon and P derived from the same virus was not sufficient for high levels of encapsidation, implying that other viral interactions contribute to encapsidation. Chimeric analysis showed that a large sequence containing region II may interact with P and/or C for efficient encapsidation. Further analysis demonstrated that possibly an RNA-RNA interaction between the intervening sequence and region II facilitates pgRNA encapsidation. Together, these results identify functional interactions among various viral components that contribute to pgRNA encapsidation.

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, and cis-acting sequences are indicated. The terminal redundancy, R, is labeled on pgRNA. For encapsidation, P binds to ɛ for the packaging of P and pgRNA, but little is known about other required interactions. The binding of P to ɛ may initiate the cooperative assembly of C subunits around pgRNA. Alternatively, capsid assembly may occur concomitantly or before the P-ɛ interaction. pgRNA encapsidation is a prerequisite for reverse transcription and virion production.

FIG. 2.

Experimental strategy for analyzing encapsidation of hepadnavirus chimeras. A test plasmid and an internal standard plasmid were cotransfected into LMH cells. This figure represents the pgRNA of DHBV and the DHBV internal standard. Nucleotide coordinates of the 5′ end, region II, and the position of the P^Y96F mutation are included. The test plasmid is the wild-type reference or the chimeric variant whose encapsidation was tested. All test plasmids are null for the synthesis of P and C proteins. The internal standard provided P^Y96F and C in trans. After three days, A and C fractions of RNA were isolated from the cells. RPA was performed to detect the test RNA and internal standard RNA in the A and C fractions. To detect the chimeric RNAs, two probes, one with DHBV sequence and one with HHBV sequence, were used simultaneously. Also, the P^Y96F substitution in the DHBV and HHBV internal standards allowed the internal standard RNA to be distinguished from the test RNA in the RPA. Encapsidation was measured by comparing the level of test RNA found in the C fraction, normalized to the internal standard in the C fraction, to the level of test RNA found in the A fraction, normalized to the internal standard in the A fraction.

FIG. 3.

DHBV P and C support efficient encapsidation of HHBV pgRNA, but HHBV P and C do not support efficient encapsidation of DHBV pgRNA. (A) RPA of DHBV and HHBV cotransfected with the DHBV internal standard. The positions of test RNA, internal standard RNA, and the probe are indicated. Lane 1, probes undigested; lane 2, probes digested; lanes 3 and 4, test RNA alone or internal standard RNA alone; lanes 5 and 7, cytoplasmic poly(A) fraction of RNA (A RNA); lanes 6 and 8, capsid fraction of RNA (C RNA). The source of the replication proteins is adjacent to ovals (P protein) and pentagons (C protein). The histogram compares the encapsidation efficiency of HHBV with that of DHBV using DHBV P and C proteins. Results are averages and standard deviations from six independent analyses. (B) RPA of HHBV and DHBV cotransfected with the HHBV internal standard. Lanes are as explained in the legend to panel A and as indicated. The histogram compares the encapsidation efficiency of DHBV with that of HHBV using HHBV P and C proteins. Results are averages and standard deviations from four independent analyses.

FIG. 4.

A DHBV with HHBV ɛ shows impaired encapsidation when HHBV P and C are used but not when DHBV P and C are used. (A) Schematic of HHBV, DHBV, and HHBV ɛ pgRNA. Thin line, DHBV sequence; thick line, HHBV sequence. Nucleotide coordinates at substitution boundaries of the chimeras represent the HHBV position. In the table on the right, the left column shows encapsidation efficiency using HHBV P and C and normalized to HHBV, while the right column shows encapsidation efficiency using DHBV P and C and normalized to DHBV. Data for the encapsidation efficiency of HHBV ɛ are averages and standard deviations from four independent analyses. (B) RPA of HHBV ɛ cotransfected with HHBV P and C and compared to HHBV. (C) RPA of HHBV ɛ cotransfected with DHBV P and C and compared to DHBV. For panels B and C, lanes are as explained in the legend to Fig. 3A and as indicated.

FIG. 5.

HHBV ɛ and nt 1 to 717 are required for efficient encapsidation using HHBV P and C. (A) Schematic of pgRNAs synthesized by test plasmids of HHBV or HHBV/DHBV chimeras and their encapsidation efficiency using HHBV P and C and normalized to HHBV. Nucleotide coordinates at substitution boundaries of the chimeras represent the HHBV position. Results are averages and standard deviations from four independent analyses for all variants, except for HHBV 1-717, results for which represent five independent analyses. (B and C) RPAs of HHBV/DHBV chimeras cotransfected with the HHBV internal standard. Lanes are as explained in the legend to Fig. 3A and as indicated.

FIG. 6.

An interaction within region II and/or the intervening sequence may contribute to encapsidation. (A) pgRNAs of DHBV and HHBV/DHBV chimeras and their encapsidation efficiency using DHBV P and C and normalized to DHBV. Nucleotide coordinates at substitution boundaries of the chimeras represent the HHBV position. Results are averages and standard deviations from four independent experiments for all chimeras except HHBV ɛ/1-717, results for which represent five independent analyses. (B and C) RPAs of HHBV/DHBV chimeras cotransfected with the DHBV internal standard. Lanes are as explained in the legend to Fig. 3A and as indicated.

FIG. 7.

Reciprocal DHBV/HHBV chimeras show that the intervening sequence needs to be compatible with region II for encapsidation. (A) Schematic of pgRNAs of reciprocal chimeras with DHBV encapsidation sequences substituted into HHBV. Nucleotide coordinates at substitution boundaries of the chimeras represent the DHBV position. These chimeras were cotransfected with the DHBV internal standard, and encapsidation efficiency was compared to that of DHBV. Results are averages and standard deviations from four independent experiments for all chimeras except DHBV RII, which was analyzed five times. (B) RPA of reciprocal chimeras cotransfected with the DHBV internal standard. Lanes are as explained in the legend to Fig. 3A and as indicated.