In vitro assembly of mosaic hepatitis B virus capsid‐like particles (CLPs): Rescue into CLPs of assembly‐deficient core protein fusions and FRET‐suited CLPs

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Duck hepatitis B virus (DHBV) shares many fundamental features with human HBV. However, the DHBV core protein (DHBc), forming the nucleocapsid shell, is much larger than that of HBV (HBc) and, in contrast to HBc, there is little direct information on its structure. Here we applied an efficient expression system for recombinant DHBc particles to the biochemical analysis of a large panel of mutant DHBc proteins. By combining these data with primary sequence alignments, secondary structure prediction, and three-dimensional modeling, we propose a model for the fold of DHBc. Its major features are a HBc-like two-domain structure with an assembly domain comprising the first about 185 amino acids and a C-terminal nucleic acid binding domain (CTD), connected by a morphogenic linker region that is longer than in HBc and extends into the CTD. The assembly domain shares with HBc a framework of four major ␣-helices but is decorated at its tip with an extra element that contains at least one helix and that is made up only in part by the previously predicted insertion sequence. All subelements are interconnected, such that structural changes at one site are transmitted to others, resulting in an unexpected variability of particle morphologies. Key features of the model are independently supported by the accompanying epitope mapping study. These data should be valuable for functional studies on the impact of core protein structure on virus replication, and some of the mutant proteins may be particularly suitable for higher-resolution structural investigations.Hepatitis B viruses (HBVs), or hepadnaviruses, comprise a family of small enveloped DNA-containing viruses that replicate through reverse transcription (2). HBV, the causative agent of B-type hepatitis in humans, is the prototype of the orthohepadnaviruses which infect selected mammals, while duck HBV (DHBV) is the prototype of the avihepadnaviruses, which are endemic in some bird species (16). Overall, genome organization and replication mechanism of both genera are closely related; hence, DHBV serves as an important model hepadnavirus (43).However, although the DHBV genome is even smaller than that of HBV (3.0 kb versus 3.2 kb), its core protein (DHBc) is substantially larger (262 versus 183 or 185 amino acids) than that of HBV (HBc). Both core proteins are the sole building blocks for the viral capsid shell. The capsids are actively involved in reverse transcription (21, 33, 55) and genome trafficking (23); are the substrate for various phosphorylation and dephosphorylation events (1,17,25,32,37,57); and provide interaction sites, regulated by the maturation state of the packaged genome (47), for envelopment by the surface proteins (9). Evidently, the short HBc sequence fully supports these multiple functions; hence, the biological reasons behind the larger size of the avihepadnavirus core proteins are enigmatic. Knowledge of the DHBc structure would be crucial to understand this unresolved issue, and it might help to exploit the experimental advantages of DHBV (43) fo...

Section: Methodsmentioning

confidence: 99%

A Structural Model for Duck Hepatitis B Virus Core Protein Derived by Extensive Mutagenesis

Nassal

Leifer

Wingert

et al. 2007

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“…As the rules for regulating and deregulating assembly are determined for different viruses, targeting assembly holds promise for a new generation of antiviral therapeutics (42). Recent development of highthroughput methods for screening potential anti-HBV capsid-specific molecules will help identify molecules that use this and other mechanisms for affecting capsid assembly and structure (28,34).…”

Section: Discussionmentioning

confidence: 99%

Global Structural Changes in Hepatitis B Virus Capsids Induced by the Assembly Effector HAP1

Bourne

Finn

Zlotnick

2006

162

272

Hepatitis B virus (HBV) is a leading cause of liver disease and hepatocellular carcinoma; over 400 million people are chronically infected with HBV. Specific anti-HBV treatments, like most antivirals, target enzymes that are similar to host proteins. Virus capsid protein has no human homolog, making its assembly a promising but undeveloped therapeutic target. HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a heteroaryldihydropyrimidine, is a potent HBV capsid assembly activator and misdirector. Knowledge of the structural basis for this activity would directly benefit the development of capsid-targeting therapeutic strategies. This report details the crystal structures of icosahedral HBV capsids with and without HAP1. We show that HAP1 leads to global structural changes by movements of subunits as connected rigid bodies. The observed movements cause the fivefold vertices to protrude from the liganded capsid, the threefold vertices to open, and the quasi-sixfold vertices to flatten, explaining the effects of HAP1 on assembled capsids and on the assembly process. We have identified a likely HAP1-binding site that bridges elements of secondary structure within a capsid-bound monomer, offering explanation for assembly activation. This site also interferes with interactions between capsid proteins, leading to quaternary changes and presumably assembly misdirection. These results demonstrate the plasticity of HBV capsids and the molecular basis for a tenable antiviral strategy. Perturbing HBV assembly, altering either the timing or the geometry of capsid formation, is expected to interfere with viral infection (42). In cultured cells, heteroaryldihydropyrimidine (HAP) molecules, such as BAY41-4109, lead to decreased production of virions and accelerated loss of capsid protein to proteasomal digestion (8). In vitro, excess BAY41-4109 led to misdirection of assembly (11). HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a related small molecule, was found to accelerate capsid assembly kinetics, while stoichiometric concentrations of HAP1 misdirect assembly, yielding nonicosahedral hexagonal arrays of capsid protein (26). HAP1 also bound preformed capsids and triggered disassembly, which was exacerbated by capsid-destabilizing conditions (26). Hepatitis B virus (HBV) isTo ascertain the structural basis for HAP1 activity, we have determined the 5.05-Å structure of a variant of the adyw strain HBV capsid (residues 1 to 149) complexed with HAP1 (referred to as ϩHAP1). To facilitate comparison, we have also determined the structure of this variant in the absence of HAP1 (ϪHAP1) to 3.95 Å. We find structural changes resulting from binding HAP1 that suggest a molecular basis for HAP1 activity. We also identify a putative HAP1-binding site, defining a target for assembly-directed anti-HBV molecules. MATERIALS AND METHODSSample preparation. Hepatitis B virus strain adyw, truncated at residue 149, had three native cyst...

“…The size of the capsid protein monomer is similar to that of a FP molecule, and the presence of a FP could interfere with the highly symmetrical packing of trimeric capsomers. One solution has been to coexpress both tagged and untagged versions of the viral protein (8). Smaller amounts of FP-tagged proteins can still be sufficient for a bright fluorescent signal, while untagged proteins provide more space for the incorporated FP fusions.…”

Section: Physical Properties Of Fluorescent Proteinsmentioning

confidence: 99%

Going Viral with Fluorescent Proteins

Costantini

Snapp

2015

Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses. Statements such as "my fluorescent virus can barely replicate" and "I cannot see my fluorescent protein fusion" are, unfortunately, not uncommon complaints about fusion proteins made with fluorescent proteins (FPs). In some cases, FPs are inappropriate for the system or question. Other times, FPs are incorporated in a haphazard manner. Finally, FPs may not perform as advertised or the investigator may not read the "fine print." In this review, we describe how to avoid common FP problems and how to select appropriate FPs for FP fusions.With multiple fluorescent labeling strategies available, why use FP tags? Generally, FPs have many desirable characteristics. They are genetically encoded, have sufficiently short sequences for incorporation into many viral genomes, and enable site-specific labeling of a viral or cellular protein of interest. Dye-labeling techniques often randomly label proteins and require extracellular delivery, and dye-labeled proteins cannot be delivered into subcellular organelles, such as the endoplasmic reticulum. Difficulties frequently arise when investigators expect FPs to be inert, well behaved in all environments, and provide a bright signal. While few, if any, FPs satisfy every item on a wish list, FPs are undeniably powerful cell biology tools. For a detailed list of virus-relevant methods that exploit FPs, see reference 1 (especially Tables 1 and 2). For basic considerations for developing FP fusion proteins, see reference 2. PHYSICAL PROPERTIES OF FLUORESCENT PROTEINSFirst, let us consider the general properties of FPs. They have short primary sequences but fold into proteins that are not small (ϳ5 nm in diameter) (3). FPs are evolved as soluble cytoplasmic proteins, with only the environmental considerations related to the pH-neutral cytoplasm as a selective pressure. Taken together, these properties suggest significant concerns when using FPs, such as the potential for steric hindrance in fusion proteins and for the performance of FPs in subcellular compartments. The photophysical characteristics of FPs range tremendously in brightness and photostability. Unless the investigator is studying isolated FPs or using advanced microscopy techniques, including total internal-reflection fluorescence (TIRF) (4) and photoactivated localization microscopy (PALM) (5), it is difficult to detect the signal of a single FP in the presence of the cellular autofluorescence background (2). The photophysical pr...