Sedimentation Velocity Analysis Research Articles

Chemical Approaches for Studying Histone Modifications

Histones form the protein core around which genomic DNA is wrapped in eukaryotic chromatin. Numerous genetic studies have established that the structure and transcriptional state of chromatin are closely related to histone post-translational modifications. Further elucidation of the precise mechanistic roles for individual histone modifications requires the ability to isolate and study homogeneously modified histones. However, the highly heterogeneous nature of histone modifications in vivo poses a significant challenge for such studies. Chemical tools that have enabled biochemical and biophysical studies of site-specifically modified histones are the focus of this minireview.

The structure of dopamine induced α-synuclein oligomers

Inclusions of aggregated alpha-synuclein (alpha-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson's disease (PD). In vitro, alpha-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of alpha-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on alpha-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive beta-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of alpha-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the beta-sheet structure found in other pre-fibrillar forms of alpha-syn.

Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 "long" (M1) and "short" (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 degrees C indicated that OAPs diffuse slowly (D approximately 10(-12) cm(2)/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.

Substrate-mediated Stabilization of a Tetrameric Drug Target Reveals Achilles Heel in Anthrax

Bacillus anthracis is a gram-positive spore-forming bacterium that causes anthrax. With the increased threat of anthrax in biowarfare, there is an urgent need to characterize new antimicrobial targets from B. anthracis. One such target is dihydrodipicolinate synthase (DHDPS), which catalyzes the committed step in the pathway yielding meso-diaminopimelate and lysine. In this study, we employed CD spectroscopy to demonstrate that the thermostability of DHDPS from B. anthracis (Ba-DHDPS) is significantly enhanced in the presence of the substrate, pyruvate. Analytical ultracentrifugation studies show that the tetramer-dimer dissociation constant of the enzyme is 3-fold tighter in the presence of pyruvate compared with the apo form. To examine the significance of this substrate-mediated stabilization phenomenon, a dimeric mutant of Ba-DHDPS (L170E/G191E) was generated and shown to have markedly reduced activity compared with the wild-type tetramer. This demonstrates that the substrate, pyruvate, stabilizes the active form of the enzyme. We next determined the high resolution (2.15 A) crystal structure of Ba-DHDPS in complex with pyruvate (3HIJ) and compared this to the apo structure (1XL9). Structural analyses show that there is a significant (91 A(2)) increase in buried surface area at the tetramerization interface of the pyruvate-bound structure. This study describes a new mechanism for stabilization of the active oligomeric form of an antibiotic target from B. anthracis and reveals an "Achilles heel" that can be exploited in structure-based drug design.

Expression of, and in vivo stressosome formation by, single members of the RsbR protein family in Bacillus subtilis

The Bacillus subtilis stressosome is a 1.8 MDa complex that is the focal point for activating the bacterium's general response to physical stress. In vitro studies demonstrated that the stressosome's core element can be formed from one or more of a family of paralogous proteins (RsbRA, -RB, -RC and -RD) onto which the system's activator protein (RsbT) and its principal inhibitor (RsbS) are bound. The RsbR components of the stressosome are envisioned to be the initial receptors of stress signalling with the stressosome structure itself serving as a device to integrate multiple stress signals for a coordinated response. In the current work, we examine several of the in vivo characteristics of the RsbR family members, including their expression and ability to form stressosomes to regulate sigma(B). Translational fusions of lacZ to each rsbR paralogue revealed that rsbRA, -RB and -RC are expressed at similar levels, which remain relatively constant during growth, ethanol stress and entry into stationary phase. rsbRD, in contrast, is expressed at a level that is only slightly above background during growth, but is induced to 30 % of the rsbRA expression level following ethanol stress. Velocity sedimentation analyses of B. subtilis extracts from strains expressing single rsbR paralogues demonstrated that each incorporates RsbS into fast-sedimenting complexes. However, consistent with rsbRD's lower expression, the RsbRD-dependent RsbS complexes were present at only 20 % of the level of the complexes seen in a wild-type strain. The lower stressosome level in the RsbRD strain is still able to hold RsbT's activity in check, implying that the RsbR/S component of stressosomes is normally in excess for the control of RsbT. Consistent with such a notion, reporter gene and Western blot assays demonstrate that although RsbT is synthesized at the same rate as RsbRA and RsbS, RsbT's ultimate level in growing B. subtilis is only 10 % that of RsbRA. Apparently, RsbT's inherent structure and/or its passage between the stressosome and its activation target compromises its persistence.

A Hydrodynamic Analysis of APOBEC3G Reveals a Monomer−Dimer−Tetramer Self-Association That Has Implications for Anti-HIV Function

The innate antiviral factor APOBEC3G (A3G) possesses RNA binding activity and deaminates HIV-1 DNA. High-molecular mass forms of A3G can be isolated from a variety of cell types but exhibit limited deaminase activity relative to low-molecular mass species prepared under RNA-depleted conditions. To investigate the fundamental oligomeric state and shape of A3G, we conducted sedimentation velocity analyses of the pure enzyme under RNA-deficient conditions. The results reveal a predominant dimer in equilibrium with minor monomeric and tetrameric species. Hydrodynamic modeling of the dimer supports an extended cylindrical shape that assembles into an elongated tetramer. Overall, the results provide physical restraints for the A3G quaternary structure that have implications for modulating antiviral function.

Scaling for Sedimentation and Diffusion of Poly(ethylene glycol) in Water

The sedimentation and diffusion of poly(ethylene glycol) (PEG) with weight average molecular weight (M(w)) from 5 x 10(2) to 2 x 10(5) g/mol have been investigated by use of an analytical ultracentrifuge (AUC). The sedimentation velocity analysis based on the Lamm equation gives the sedimentation coefficient (s) and diffusion coefficient (D). Our study reveals that the sedimentation coefficient (s(0)) and diffusion coefficient (D(0)) at infinite dilution scale to molecular weight (M(w)), namely, s(0) = K(s)M(w)(alpha), with K(s) = 6.14 x 10(-3) and alpha = 0.469 +/- 0.008, and D(0) = K(D)M(w)(-beta), with K(D) = 1.43 x 10(-8) and beta = 0.576 +/- 0.007.

Conformational Properties of β-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

Oligomeric Structure of Colicin Ia Channel in Lipid Bilayer Membranes

Colicin Ia is a soluble, harpoon-shaped bacteriocin which translocates across the periplasmic space of sensitive Escherichia coli cell by parasitizing an outer membrane receptor and forms voltage-gated ion channels in the inner membrane. This process leads to cell death, which has been thought to be caused by a single colicin Ia molecule. To directly visualize the three-dimensional structure of the channel, we generated two-dimensional crystals of colicin Ia inserted in lipid-bilayer membranes and determined a approximately 17 three-dimensional model by electron crystallography. Supported by velocity sedimentation, chemical cross-linking and single-particle image analysis, the three-dimensional structure is a crown-shaped oligomer enclosing a approximately 35 A-wide extrabilayer vestibule. Our study suggests that lipid insertion instigates a global conformational change in colicin Ia and that more than one molecule participates in the channel architecture with the vestibule, possibly facilitating the known large scale peptide translocation upon channel opening.

Dimeric Quaternary Structure of the Prototypical Dual Specificity Phosphatase VH1

The Vaccinia virus H1 gene product, VH1, is a dual specificity phosphatase that down-regulates the cellular antiviral response by dephosphorylating STAT1. The crystal structure of VH1, determined at 1.32 A resolution, reveals a novel dimeric quaternary structure, which exposes two active sites spaced approximately 39 A away from each other. VH1 forms a stable dimer via an extensive domain swap of the N-terminal helix (residues 1-20). In vitro, VH1 can dephosphorylate activated STAT1, in a reaction that is competed by the nuclear transport adapter importin alpha5. Interestingly, VH1 is inactive with respect to STAT1 bound to DNA, suggesting that the viral phosphatase acts predominantly on the cytoplasmic pool of activated STAT1. We propose that the dimeric quaternary structure of VH1 is essential for specific recognition of activated STAT1, which prevents its nuclear translocation, thus blocking interferon-gamma signal transduction and antiviral response.

Phenomenological partial-specific volumes for G-quadruplex DNAs.

Accurate partial-specific volume ([Formula: see text]) values are required for sedimentation velocity and sedimentation equilibrium analyses. For nucleic acids, the estimation of these values is complicated by the fact that [Formula: see text] depends on base composition, secondary structure, solvation and the concentrations and identities of ions in the surrounding buffer. Here we describe sedimentation equilibrium measurements of the apparent isopotential partial-specific volume /' for two G-quadruplex DNAs and a single-stranded DNA of similar molecular weight and base composition. The G-quadruplex DNAs are a 22 nucleotide fragment of the human telomere consensus sequence and a 27 nucleotide fragment from the human c-myc promoter. The single-stranded DNA is 26 nucleotides long and is designed to have low propensity to form secondary structures. Parallel measurements were made in buffers containing NaCl and in buffers containing KCl, spanning the range 0.09 M <or= [salt] <or= 2.3 M. Limiting values of /', extrapolated to [salt] = 0 M, were: 22-mer (NaCl-form), 0.525 +/- 0.004 mL/g; 22-mer (KCl-form), 0.531 +/- 0.006 mL/g; 27-mer (NaCl-form), 0.548 +/- 0.005 mL/g; 27-mer (KCl-form), 0.557 +/- 0.006 mL/g; 26-mer (NaCl-form), 0.555 +/- 0.004 mL/g; 26-mer (KCl-form), 0.564 +/- 0.006 mL/g. Small changes /' with [salt] suggest that large changes in counterion association or hydration are unlikely to take place over these concentration ranges.

Fluorescence Detection of a Lipid-induced Tetrameric Intermediate in Amyloid Fibril Formation by Apolipoprotein C-II

The misfolding and self-assembly of proteins into amyloid fibrils that occurs in several debilitating and age-related diseases is affected by common components of amyloid deposits, notably lipids and lipid complexes. We have examined the effect of the short-chain phospholipids, dihexanoylphosphatidylcholine (DHPC) and dihexanoylphosphatidylserine (DHPS), on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Micellar DHPC and DHPS strongly inhibited apoC-II fibril formation, whereas submicellar levels of these lipids accelerated apoC-II fibril formation to a similar degree. These results indicate that the net negative charge on DHPS, compared with the neutrally charged DHPC, is not critical for either the inhibition or activation process. We also investigated the mechanism for the submicellar, lipid-induced activation of fibril formation. Emission data for fluorescently labeled apoC-II indicated that DHPC and DHPS stimulate the early formation and accumulation of oligomeric species. Sedimentation velocity and equilibrium experiments using a new fluorescence detection system identified a discrete lipid-induced tetramer formed at low apoC-II concentrations in the absence of significant fibril formation. Seeding experiments showed that this tetramer was on the fibril-forming pathway. Fluorescence resonance energy transfer experiments established that this tetramer forms rapidly and is stabilized by submicellar, but not micellar, concentrations of DHPC and DHPS. Several recent studies show that oligomeric intermediates in amyloid fibril formation are toxic. Our results indicate that lipids promote on-pathway intermediates of apoC-II fibril assembly and that the accumulation of a discrete tetrameric intermediate depends on the molecular state of the lipid.

Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+–ATPase as an example

For structural studies of integral membrane proteins, including their 3D crystallization, the judicious use of detergent for solubilization and purification is required. Detergent binding by the solubilized protein is an important parameter to determine the hydrodynamic properties in terms of size and aggregational (monomeric/oligo(proto)meric) state of the protein. Detergent binding can be measured by gel filtration chromatography under equilibrium conditions and after separation from mixed micelles of solubilized lipid and detergent. Using sarcoplasmic reticulum Ca(2+)-ATPase as an example, we demonstrate in this protocol complete procedures for measurement of detergent binding using (i) radiolabeled n-dodecyl-beta-D-maltoside (DM) or (ii) from measurements of the increase in refractive index due to the presence of bound detergent on the protein. The latter measurement can also be performed by sedimentation velocity (SV) analysis in the analytical ultracentrifuge which in addition allows determination of the sedimentation coefficient. In combination with estimation of Stokes radius by gel filtration calibration, the molecular mass and asymmetry of the solubilized protein can be calculated. In the proposed protocols, the gel chromatographic procedures require 1 d; SV experiments are performed just after size exclusion. The whole time for these experiments is 24 h. Data analysis of analytical ultracentrifugation requires a couple of days.

Structure and Evolution of a Novel Dimeric Enzyme from a Clinically Important Bacterial Pathogen

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine biosynthetic pathway. The tetrameric structure of DHDPS is thought to be essential for enzymatic activity, as isolated dimeric mutants of Escherichia coli DHDPS possess less than 2.5% that of the activity of the wild-type tetramer. It has recently been proposed that the dimeric form lacks activity due to increased dynamics. Tetramerization, by buttressing two dimers together, reduces dynamics in the dimeric unit and explains why all active bacterial DHDPS enzymes to date have been shown to be homo-tetrameric. However, in this study we demonstrate for the first time that DHDPS from methicillin-resistant Staphylococcus aureus (MRSA) exists in a monomer-dimer equilibrium in solution. Fluorescence-detected analytical ultracentrifugation was employed to show that the dimerization dissociation constant of MRSA-DHDPS is 33 nm in the absence of substrates and 29 nm in the presence of (S)-aspartate semialdehyde (ASA), but is 20-fold tighter in the presence of the substrate pyruvate (1.6 nm). The MRSA-DHDPS dimer exhibits a ping-pong kinetic mechanism (k(cat)=70+/-2 s(-1), K(m)(Pyruvate)=0.11+/-0.01 mm, and K(m)(ASA)=0.22+/-0.02 mm) and shows ASA substrate inhibition with a K(si)(ASA) of 2.7+/-0.9 mm. We also demonstrate that unlike the E. coli tetramer, the MRSA-DHDPS dimer is insensitive to lysine inhibition. The near atomic resolution (1.45 A) crystal structure confirms the dimeric quaternary structure and reveals that the dimerization interface of the MRSA enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species. These data provide a detailed mechanistic insight into DHDPS catalysis and the evolution of quaternary structure of this important bacterial enzyme.

Crystal Structure of Human Edc3 and Its Functional Implications

Edc3 is an enhancer of decapping and serves as a scaffold that aggregates mRNA ribonucleoproteins together for P-body formation. Edc3 forms a network of interactions with the components of the mRNA decapping machinery and has a modular domain architecture consisting of an N-terminal Lsm domain, a central FDF domain, and a C-terminal YjeF-N domain. We have determined the crystal structure of the N-terminally truncated human Edc3 at a resolution of 2.2 A. The structure reveals that the YjeF-N domain of Edc3 possesses a divergent Rossmann fold topology that forms a dimer, which is supported by sedimentation velocity and sedimentation equilibrium analysis in solution. The dimerization interface of Edc3 is highly conserved in eukaryotes despite the overall low sequence homology across species. Structure-based site-directed mutagenesis revealed dimerization is required for efficient RNA binding, P-body formation, and likely for regulating the yeast Rps28B mRNA as well, suggesting that the dimeric form of Edc3 is a structural and functional unit in mRNA degradation.

Human Regulatory Protein Ki-1/57 Has Characteristics of an Intrinsically Unstructured Protein

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1, in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Small-angle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein. Experimental curves together with ab initio modeling approaches revealed an extended and flexible molecule in solution. An elongated shape was also observed by analytical gel filtration. Furthermore, sedimentation velocity analysis suggested that Ki-1/57 is a highly asymmetric protein. These findings may explain the functional plasticity of Ki-1/57, as suggested by the wide array of proteins with which it is capable of interacting in yeast two-hybrid interaction assays.

The Complex Structure Transition of Humanin Peptides by Sodium Dodecylsulfate and Trifluoroethanol

We have examined the structure of two Humanin (HN) analog peptides, HNG and AGA-(C8R)HNG17, in the presence of sodium dodecylsulfate (SDS) and trifluoroethanol (TFE) using CD and sedimentation velocity. Both HNG and AGA-(C8R)HNG17 underwent complex conformational changes with increasing concentrations of SDS and TFE, in contrast to general trend of increasing alpha-helix with their concentration. To our surprise, both peptides appear to converge into a similar structure in SDS and TFE at higher concentrations; e.g., above 0.05 % SDS or 30-40 % TFE. Sedimentation velocity analysis showed extensive aggregation of HNG at 0.1 mg/ml in PBS in the absence of SDS, but a highly homogeneous solution in 0.1 % SDS, indicating formation of a uniform structure by SDS. These two peptides also formed an intermediate structure both in SDS and TFE at lower concentrations, which appeared to be associated with extensive aggregation. It is interesting that the structure changes of these peptides occur well below the critical micelle concentration of SDS, suggesting that conformational changes are mediated through molecular, not micellar, interactions with SDS.

Analysis of intermolecular base pair formation of prohead RNA of the phage ø29 DNA packaging motor using NMR spectroscopy

The bacteriophage ø29 DNA packaging motor that assembles on the precursor capsid (prohead) contains an essential 174-nt structural RNA (pRNA) that forms multimers. To determine the structural features of the CE- and D-loops believed to be involved in multimerization of pRNA, 35- and 19-nt RNA molecules containing the CE-loop or the D-loop, respectively, were produced and shown to form a heterodimer in a Mg2+-dependent manner, similar to that with full-length pRNA. It has been hypothesized that four intermolecular base pairs are formed between pRNA molecules. Our NMR study of the heterodimer, for the first time, proved directly the existence of two intermolecular Watson–Crick G–C base pairs. The two potential intermolecular A–U base pairs were not observed. In addition, flexibility of the D-loop was found to be important since a Watson–Crick base pair introduced at the base of the D-loop disrupted the formation of the intermolecular G–C hydrogen bonds, and therefore affected heterodimerization. Introduction of this mutation into the biologically active 120-nt pRNA (U80C mutant) resulted in no detectable dimerization at ambient temperature as shown by native gel and sedimentation velocity analyses. Interestingly, this pRNA bound to prohead and packaged DNA as well as the wild-type 120-nt pRNA.

Structural Basis for the Recognition and Cross-linking of Amyloid Fibrils by Human Apolipoprotein E

Apolipoprotein (apo) E is a well characterized lipid-binding protein in plasma that also exists as a common nonfibrillar component of both cerebral and systemic amyloid deposits. A genetic link between a common isoform of apoE, apoE4, and the incidence of late onset Alzheimer disease has drawn considerable attention to the potential roles of apoE in amyloid-related disease. We examined the interactions of apoE with amyloid fibrils composed of apoC-II and the amyloid-beta (Abeta) peptide. Aggregates of apoE with Abeta and apoC-II are found in Alzheimer and atherosclerotic plaques, respectively. Sedimentation velocity and fibril size distribution analysis showed that apoE3 and E4 isoforms bind and noncovalently cross-link apoC-II fibrils in a similar manner. This ability to cross-link apoC-II fibrils was abolished by the dissociation of the apoE tetramer to monomers or by thrombin cleavage to yield separate N- and C-terminal domains. Preparative ultracentrifuge binding studies indicated that apoE and the isolated N- and C-terminal domains of apoE bind with submicromolar affinities to both apoC-II and Abeta fibrils. Fluorescence quenching and resonance energy transfer experiments confirmed that both domains of apoE interact with apoC-II fibrils and demonstrated that the binding of the isolated N-terminal domain of apoE to apoC-II or Abeta fibrils is accompanied by a significant conformational change with helix three of the domain moving relative to helix one. We propose a model involving the interaction of apoE with patterns of aligned residues that could explain the general ability of apoE to bind to a diverse range of amyloid fibrils.

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.