1 | Integration of Hepatitis B Virus (HBV) DNA: Underlying mechanisms, its role in pathogenesis, and its uses as a clinical marker

Inte­gra­tion of Hepati­tis B Virus (HBV) DNA into the host genome occurs in all known mem­bers of the hepad­naviri­dae fam­i­ly, despite this form not being nec­es­sary for the for­ma­tion of new viri­ons. How HBV DNA inte­gra­tion occurs and its func­tion is com­plete­ly unknown. Of clin­i­cal rel­e­vance, HBV DNA inte­gra­tion is asso­ci­at­ed liv­er can­cer, with the major­i­ty of HBV-asso­ci­at­ed hepa­to­cel­lu­lar car­ci­no­ma (HCC) con­tain­ing one or more HBV DNA inte­gra­tions. This is a dra­mat­i­cal­ly high­er fre­quen­cy com­pared to the 1 inter­gra­tion per ~103–104 cells in the gen­er­al hepa­to­cyte population.

In recent­ly pub­lished work, we have devel­oped and char­ac­terised an in vit­ro sys­tem to reli­ably detect HBV DNA inte­gra­tions resul­tant from a bona fide HBV infec­tion. Using this sys­tem and oth­er devel­oped tech­niques, we intend to deter­mine: i) how HBV DNA inte­grates; ii) what the con­se­quences of this inte­gra­tion are (par­tic­u­lar­ly in hepa­to­car­cino­gen­e­sis); and iii) if these inte­gra­tion events gen­er­ate detectable bio­mark­ers that can pre­dict liv­er can­cer risk.

2 | Investigation of the mechanism of innate immunity activation by HDV replication and the role of IFN response in HDV chronicity

In con­trast to HBV, HDV infec­tion induces pro­found innate immune response which is medi­at­ed by pat­tern recog­ni­tion recep­tor MDA5 (Zhang, et al. 2018. J Hepa­tol. 69:25–35). MDA5 is a cytosol sen­sor usu­al­ly rec­og­niz­ing long dou­ble strand RNA (dsR­NA). How­ev­er, unlike oth­er RNA virus­es, HDV repli­cates its RNA in the nucle­us via a unique dou­ble-rolling-cycle mech­a­nism with­out pro­duc­ing long dsR­NA. Inves­ti­gat­ing the innate immune acti­va­tion dur­ing HDV repli­ca­tion will pro­vide impor­tant insights for under­stand­ing MDA5 medi­at­ed innate immune sens­ing of viral RNA. To this aim, we plan to: (i) iden­ti­fy the HDV RNA lig­and rec­og­nized by MDA5; (ii) deter­mine the sub­cel­lu­lar loca­tion of MDA5-HDV RNA inter­ac­tion; and (iii) elu­ci­date the roles of host fac­tors (LGP2, ADAR1, etc.) and viral fac­tors (HDAg and HBV enve­lope pro­teins) in the process of innate immu­ni­ty activation.

Long term per­sis­tence of HBV and HDV makes it chal­leng­ing to devel­op cura­tive ther­a­pies. Extra­cel­lu­lar spread­ing path­ways are impor­tant for per­sis­tence of both virus­es and can be blocked by the entry inhibitor Myr­cludex B. How­ev­er, HDV can also prop­a­gate through cell divi­sion. Our work has proved that this cell divi­sion medi­at­ed HDV spread is very sen­si­tive to IFN response (unpub­lished).  In this project we aim to elu­ci­date the mech­a­nism of cell divi­sion medi­at­ed HDV spread with the focus of under­stand­ing the role of IFN response in this process. To this end, we intend to: (i) char­ac­ter­ize cell divi­sion medi­at­ed HDV spread in dif­fer­ent cell lines with or with­out IFN response; (ii) iden­ti­fy the IFN induced antivi­ral fac­tors con­tribut­ing the block­ade of HDV spread dur­ing cell divi­sion; (iii) estab­lish an in vit­ro mod­el sup­port­ing both extra­cel­lu­lar and cell divi­sion medi­at­ed HDV spread; and (iv) devel­op nov­el syn­er­gis­tic co-treat­ments by comb­ing inhibitors tar­get­ing tar­get­ing extra­cel­lu­lar HDV spread (MyrB, Lon­a­farnib, etc.) and cell divi­sion medi­at­ed HDV spread (IFN, TLR ago­nists, etc.).

3 | Click labeling of HBV/HDV and the entry receptor NTCP

The incor­po­ra­tion of flu­o­res­cent pro­teins or small­er pep­tide tags into HBV pro­teins is very chal­leng­ing due to the small genome size, the over­lap­ping genes and the tight inter­ac­tions between not only the cap­sid monomers but also between cap­sid and enve­lope pro­teins, which is required for the assem­bly of the viri­ons. Genet­ic code expan­sion in com­bi­na­tion with click chem­istry is an ele­gant label­ing method, espe­cial­ly suit­ed for virus­es due to above-men­tioned rea­sons. This tech­nol­o­gy uti­lizes the pyrroly­sine tRNA/aminoa­cyl-tRNA syn­thetase (RS) pair from Methanosarci­na mazei, which is orthog­o­nal to the eukary­ot­ic trans­la­tion machin­ery and was engi­neered to enable the incor­po­ra­tion of non-canon­i­cal amino acids (ncAA) at the amber stop codon UAG. By uti­liz­ing this tech­nol­o­gy, we intro­duced ncAA into the core pro­tein of HBV, the delta anti­gen of HDV and the entry recep­tor sodi­um tau­ro­cholate cotrans­port­ing polypep­tide (NTCP), which are sub­se­quent­ly click labeled with func­tion­al­ized flu­o­rophores. Since the click label­ing we uti­lize is cop­per-inde­pen­dent, it is also com­pat­i­ble with live-cell imag­ing. In this project, we aim to cre­ate flu­o­res­cent tools to visu­al­ize not only the viri­ons but also their entry recep­tor to study (i) traf­fick­ing of NTCP with or with­out the flu­o­res­cent­ly labeled preS1 pep­tide MyrB, (ii) ear­ly entry events of HBV and HDV, (iii) intra­cel­lu­lar trans­port of HBV nucle­o­cap­sids (HBV-NC) and HDV ribonu­cle­o­pro­tein com­plex­es (HDV-RNP) on their way to the nucle­us, and (iv) intra­cel­lu­lar traf­fick­ing of de novo formed HBV-NC and HDV-RNP.