Enhancement of hepatitis B virus infection by noninfectious subviral particles

Abstract

The biological function of the huge excess of subviral particles over virions in hepatitis B virus infections is unknown. Using the duck hepatitis B virus as a model, we unexpectedly found that subviral particles strongly enhance intracellular viral replication and gene expression. This effect is dependent on the multiplicity of infection, the ratio of virions over subviral particles, and the time point of addition of subviral particles. Most importantly, we show that the pre-S protein of the subviral particles triggers enhancement and requires the presence of the binding regions for putative cell-encoded virus receptor proteins. These data suggest that enhancement is due either to the recently described transactivation function of the pre-S protein or to signalling pathways which become activated upon binding of subviral particles to cellular receptors. The findings are of clinical importance, since they imply that infectivity of sera containing hepadnaviruses depends not only on the amount of infectious virions but also decisively on the number of particles devoid of nucleic acids. A similarly dramatic enhancing effect of noninfectious particles in other virus infections is well conceivable.

FIG. 1

Separation of DHBV DNA-containing particles and SVPs by two Urografin-gradient ultracentrifugations. (a) DHBV DNA dot blot (top) and pre-S immunoblot (bottom) performed with fractions of DHBV virions and SVPs separated by centrifugation through a Urografin-gradient (0 to 40%) at 38,000 rpm for 18 h. The viral DNA-containing particles are present mainly in fractions 5 to 7, whereas SVPs are concentrated in fractions 8 and 9, as evident from the locations of the viral DNA and pre-S proteins. (c) DHBV DNA dot blot (top) and pre-S immunoblot (bottom) performed with fractions obtained by mixing partially purified virions from fractions 5 to 7 of the first gradient with 26.5% Urografin and ultracentrifugation. Viral DNA-containing particles are present mainly in fractions 3 and 4, as evident from the DNA dot blot. Pre-S protein of particles without or with only very little DNA is present in fractions 1 to 12. The amount of proteins in the fractions of this gradient was too small for visualization by silver staining when 50% of each fraction was analyzed on a SDS-polyacrylamide gel (data not shown). A nonlinear shallow gradient is formed during centrifugation. (e) DHBV DNA dot blot (top) and pre-S immunoblot (bottom) performed with fractions obtained by mixing partially purified SVPs from fractions 8 and 9 of the first gradient with 26.5% Urografin and ultracentrifugation. Silver staining of the proteins separated on an SDS-polyacrylamide gel run in parallel (50% of each fraction was loaded) revealed proteins only in fractions 13 to 16 (small panel). In these fractions, the small envelope and pre-S protein are major bands. Another major band migrating at 70 kDa reacted with antibodies to heat shock protein 70 (HSC70) on an immunoblot (data not shown). Consistent with and extending recently reported data for HBV and DHBV (38), this observation indicates that HSC70 is a major component of DHBV SVPs. (b, d, and f) Quantification of the amount of DHBV DNA and total protein in the fractions of all three gradients. LI, protein extract from the liver of an infected duck used as a positive control in the immunoblot.

FIG. 2

Dose-dependent enhancement or inhibition of DHBV infection by SVPs. (a) Detection of pre-S proteins (P36 and P28) by immunoblotting with extracts of cells incubated with duck serum containing either no DHBV particles (lane 10), various amounts of live virus including SVPs (lanes 1 to 9), or UV-inactivated virus particles, designated SVPs (lanes 2, 3, 5, 6, 8, and 9). Enhancement of infection by SVPs is seen at low but not high MOI. (b) Pre-S immunoblot with extracts from cells infected with a DHBV-positive or -negative serum (lanes 1 to 4 and 5 to 8, respectively) and various amounts of UV-irradiated, partially purified SVPs, both added simultaneously. Inhibition of DHBV infection by SVPs is observed when 10⁶ SVPs/cell are added, whereas gradual enhancement is seen with 10- and 100-fold fewer SVPs. SVPs alone do not lead to detectable levels of intracellular pre-S proteins (lanes 5 to 8).

FIG. 3

Time dependence of infection enhancement by SVPs. Southern blot analysis of replicative intermediates reveals strong enhancement of infection by highly purified SVPs only when coincubated with purified virions or when added after infection. Cells were either mock infected (lanes 1 and 8) or infected with 0.1 DHBV virion/cell and either with (lanes +) or without (lanes −) 10² SVPs/cell. SVPs were either added before (lanes 4 to 7), simultaneously with (lanes 9 and 10), or after (lanes 11 to 14) infection. The positions of relaxed-circular (RC) and single-stranded (SS) DHBV DNA are indicated.

FIG. 4

Effect of UV-irradiated virus particles of natural DHBV isolates and of pre-S mutants on DHBV infection. (a) Virus particles were first produced by transfection of LMH chicken hepatoma cells with DHBV DNA cloned from an infected duck (WT3T) or goose (WT1T) and mutants of DHBV-26 with various deletions (amino acids deleted are in parentheses) introduced into the pre-S region. (b and c) The virus particles from the culture medium, harvested 3 days after transfection, were concentrated by being pelleted through a sucrose cushion and were UV irradiated before use. Similar numbers of viral particles were applied in each experiment (0.1 virion and 100 SVPs/cell), as deduced from the presence of similar amounts of DHBV DNA and pre-S proteins detected by DNA hybridization and immunoblotting, respectively (data not shown). The LMH cell-derived SVPs were mixed with highly purified DHBV virions (0.01 genome equivalent/cell) and used for infection as described in the legend Fig. 2. Intracellular pre-S in the infected hepatocytes was detected by immunoblotting (b), and the amount of replicative intermediates was detected by Southern blotting (c).

FIG. 4

Effect of UV-irradiated virus particles of natural DHBV isolates and of pre-S mutants on DHBV infection. (a) Virus particles were first produced by transfection of LMH chicken hepatoma cells with DHBV DNA cloned from an infected duck (WT3T) or goose (WT1T) and mutants of DHBV-26 with various deletions (amino acids deleted are in parentheses) introduced into the pre-S region. (b and c) The virus particles from the culture medium, harvested 3 days after transfection, were concentrated by being pelleted through a sucrose cushion and were UV irradiated before use. Similar numbers of viral particles were applied in each experiment (0.1 virion and 100 SVPs/cell), as deduced from the presence of similar amounts of DHBV DNA and pre-S proteins detected by DNA hybridization and immunoblotting, respectively (data not shown). The LMH cell-derived SVPs were mixed with highly purified DHBV virions (0.01 genome equivalent/cell) and used for infection as described in the legend Fig. 2. Intracellular pre-S in the infected hepatocytes was detected by immunoblotting (b), and the amount of replicative intermediates was detected by Southern blotting (c).