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In vitro virus assembly

In vitro virus assembly


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Are researchers able to assemble viruses in vitro?

For example, I imagine that a phage display library may be generated by throwing in a test tube the capsid proteins (or what have you) along with relevant DNA that will self assemble. Has this been done? If so, is it a technique readily applicable to a broad range of viruses?


Yes, and a quick search for viral in-vitro assembly will turn up tons of results. - it's a little more complicated than that! - but it's not usually done as such, which I will explain. Basically, there are four things you might want to do:

  1. You want to make large amounts of virus and study it. If so, then all you need to do is infect your cell culture with your virus and let it replicate. Some are obviously easier than others - from my own experience, HIV is pretty easy but Hepatitis C is a real PITA - but that's essentially it. This obviously presumes you already have some virus stock on hand for infection. You could also transfect the culture with full-length viral plasmids, which leads me to…
  2. You want to study some specific aspect of the virus. That's cool! Most people aren't just concocting virus. Generally, for viruses that humans can get, it's generally recommended/heavily encouraged to only use what you need for safety reasons. So, for example, you might transfect the one protein you think is important to see what it does by itself. After that, you might transfect the cell culture with the full virus, but with one important gene knocked-out. I used to make single-round HIV by using a plasmid for full-length HIV without a functioning envelope gene and another plasmid for VSV envelope; new HIV particles would be made infectious because of the VSV gene but after infecting they no longer had that gene anymore. Basically, a limited version of the virus, which leads me to…
  3. You want to make virus-like particles. These are one of the better-named things in science and are just what they sound like - particles that look a lot like virus, but aren't. Generally, they're empty shells. We can use these for all sorts of things, from vaccine studies to other genes and diseases. Techniques can vary, but generally it's similar to the above.
  4. You want to introduce some other gene to a cell culture system, using a virus backbone for transduction; think "I want to fix these cells… permanently." Generally, these viruses are usually based on adenovirus or lentivirus (aka HIV) because they will insert their genes into the cell's genome, so the gene will persist.

Item 4 is probably closest to what you specifically meant. Here's a free review on the use and safety of retroviral vectors in humans, and here's a nifty one in fish. As for which viruses it's applicable to, well, that varies a lot by virus, host, culture system, and so on.


In Vitro Assembly of Retroviruses

Assembly, part of the late stages of the retroviral life cycle, begins when the structural polyprotein Gag associates with viral genomic RNA. Ultimately, more than a thousand Gag molecules form a spherical immature virion. Maturation takes place soon after or concomitantly with virus budding and is initiated as Gag is cleaved by the retroviral protease into its constituent protein domains. The immature core is thought to disassemble and the liberated CA proteins to reassemble into a morphologically distinct mature capsid. In vitro assembly with derivatives of Gag and CA has been used to study retroviruses for over two decades. In this review, we examine the discovery and development of three major model systems [human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Mason–Pfizer monkey virus (MPMV)] and discuss structural features and aspects of the retroviral assembly pathway that have been uncovered using in vitro assembly. We also put forward two major unresolved questions in the field and propose future avenues of research.


Abstract

Viruses are highly ordered supramolecular complexes that have evolved to propagate by hijacking the host cell's machinery. Although viruses are very diverse, spreading through cells of all kingdoms of life, they share common functions and properties. Next to the general interest in virology, fundamental viral mechanisms are of growing importance in other disciplines such as biomedicine and (bio)nanotechnology. However, in order to optimally make use of viruses and virus-like particles, for instance as vehicle for targeted drug delivery or as building blocks in electronics, it is essential to understand their basic chemical and physical properties and characteristics. In this context, the number of studies addressing the mechanisms governing viral properties and processes has recently grown drastically. This review summarizes a specific part of these scientific achievements, particularly addressing physical virology approaches aimed to understand the self-assembly of viruses and the mechanical properties of viral particles. Using a physicochemical perspective, we have focused on fundamental studies providing an overview of the molecular basis governing these key aspects of viral systems.

  • Biology-Inspired Nanomaterials > Protein and Virus-Based Structures
  • Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

In vitro virus assembly - Biology

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Readings

During the first class session, the instructors and the students will introduce themselves. The instructors will introduce the course and go over requirements and expectations outlined in the syllabus. The instructors then will present a brief lecture outlining the background and basics of virus structure and assembly. Finally, the topic of week 2 will be introduced.

Principles of Virus Symmetry

Watson and Crick noticed that viral nucleic acid couldn't encode a single protein big enough to enclose a virus and suggested that multiple, identical subunits polymerized into a structure to house viral nucleic acid. Caspar and Klug proposed a geometric formalism, called quasi-equivalence, to explain how 1) a spherical capsid can be built from pentamers and hexamers and 2) a protein can participate in non-equivalent interactions.

Caspar, D. L., and A. Klug. "Physical principles in the construction of regular viruses." Cold Spring Harb Symp Quant Biol 27 (1962): 1-24.

Liddington, R. C., Y. Yan, J. Moulai, R. Sahli, T. L. Benjamin, and S. C. Harrison. "Structure of simian virus 40 at 3.8-A resolution." Nature 354 (1991): 278-84.

Virus Crystallography and Cryo-EM

Crystallography, and more recently, cryoelectron microscopy, are techniques used to determine the structures of viruses. At the time that Wikoff and co-workers solved the structure of a whole virus, HK97, it was the largest structure ever solved by crystallography. Cryo-EM, though not as high resolution, provides information from frozen hydrated specimens without the need for crystals. Wu et al. determined the structure of a herpes virus and showed the locations of the subunits within the structure.

Wikoff, W. R., L. Liljas, R. L. Duda, H. Tsuruta, R. W. Hendrix, and J. E. Johnson. "Topologically linked protein rings in the bacteriophage HK97 capsid." Science 289 (2000): 2129-33.

Wu, L., P. Lo, X. Yu, J. K. Stoops, B. Forghani, and Z. H. Zhou. "Three-dimensional structure of the human herpesvirus 8 capsid." J Virol 74 (2000): 9646-54.

Capsids must be built from their component pieces. They form without any external help or energy in a process often called "self-assembly." Prevelige and King developed one of the earliest in vitro assembly systems for an icosahedral virus. Conway and co-workers used cryo-em to study the conformations of virus capsids as the matured over time, a process that results in the expansion and increased rigidity of the viral shell.

Conway, J. F., R. L. Duda, N. Cheng, R. W. Hendrix, and A. C. Steven. "Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system." J Mol Biol 253 (1995): 86-99.

Prevelige, P. E., Jr., D. Thomas, and J. A. King. "Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells." Biophys J 64, no. 3 (1993): 824-35.

Extending the principles described by Caspar and Klug, Ganser et al. propose a fullerene cone model for the HIV core. They later prove their model using an advanced cryo-EM technique called cryotomography.

Ganser, B. K., S. Li, V. Y. Klishko, J. T. Finch, and W. I. Sundquist. "Assembly and analysis of conical models for the HIV-1 core." Science 283 (1999): 80-3.

Benjamin, J., B. K. Ganser-Pornillos, W. F. Tivol, W. I. Sundquist, and G. J. Jensen. "Three-dimensional structure of HIV-1 virus-like particles by electron cryotomography." J Mol Biol 346 (2005): 577-88.

Viruses have evolved different strategies for getting their nucleic acids into shells. Bacteriophages typically use a preformed protein shell, called a procapsid, into which the DNA genome is pumped by a complex of proteins called portal and terminase. Using optical tweezers, Smith et al. show that one such packaging motor is incredibly strong. Retroviruses have entirely different mechanisms based on interactions between RNA secondary structure and the proteins that make up the surrounding shell. These protein-nucleic acid interactions are an essential component of the simultaneous assembly and packaging of a retrovirus and its genome.

D'Souza, V., and M. F. Summers. "Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus." Nature 431, no. 7008 (2004): 586-90.

Smith, D. E., S. J. Tans, S. B. Smith, S. Grimes, D. L. Anderson, and C. Bustamante. "The bacteriophage straight phi29 portal motor can package DNA against a large internal force." Nature 413 (2001): 748-52.

Virus Recognition and Attachment

The first step in a viral infection is the recognition of and attachment to a host cell. The bacteriophage P22 uses a tailspike protein that specifically recognizes and cleaves lipopolysaccharide on the surface of its host, Salmonella typhimurium, enabling it to "burrow" to the cell surface. Steinbacher and collegues determined the structure of the protein in complex with its substrate, revealing the molecular basis for the specificity. Attachment by the dengue viruses is by an equally elegant method. Unlike tailspike, the proteins that accomplish this task undergo major structural rearrangement upon interacting with the host. This restructuring of the protein triggers membrane fusion, a critical step in the infection process.

Modis, Y., S. Ogata, D. Clements, and S. C. Harrison. "Structure of the dengue virus envelope protein after membrane fusion." Nature 427, no. 6972 (2004): 313-9.

Steinbacher, S., U. Baxa, S. Miller, A. Weintraub, R. Seckler, and R. Huber. "Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors." Proc Natl Acad Sci U S A 93, no. 20 (1996): 10584-8.

Once viruses attach to their hosts, they must get their nucleic acid inside the cell. Again, viruses have evolved very diverse solutions to this problem. Poliovirus, a picornavirus, uses a peptide that binds to membranes and induces the cell to internalize the particle through the endocytotic pathway. Bacteriophage T4 instead creates a channel across its host's membranes and releases its DNA into the cell. Petr Leiman and coworkers used cryo-EM to reveal the remarkable structural changes that accompany the protein complexes of the T4 tail that accomplish this feat.

Bubeck, D., D. J. Filman, N. Cheng, A. C. Steven, J. M. Hogle, and D. M. Belnap. "The structure of the poliovirus 135S cell entry intermediate at 10-angstrom resolution reveals the location of an externalized polypeptide that binds to membranes." J Virol 79, no. 12 (2005): 7745-55.

Leiman, P. G., P. R. Chipman, V. A. Kostyuchenko, V. V. Mesyanzhinov, and M. G. Rossmann. "Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host." Cell 118, no. 4 (2004): 419-29.

Virus Structures in the Cell

Viruses are molecular freeloaders and will utilize whatever cell pathways they can. T4 capsid protein requires a chaperone to fold, but the host chaperone is too small. How does T4 deal with this problem? It has a gene encoding a chaperone cap that interacts with the host chaperone, creating a larger space in which to fit the T4 capsid protein for folding. African Swine Fever Virus uses a poorly understood cellular mechanism to assist in the folding and assembly of its capsids: the aggresome. Believed to be a cellular mechanism for dealing with misfolded proteins, these intracellular structures are induced during ASFV infection and are the sites of capsid assembly.

Bakkes, P. J., B. W. Faber, H. van Heerikhuizen, and S. M. van der Vies. "The T4-encoded cochaperonin, gp31, has unique properties that explain its requirement for the folding of the T4 major capsid protein." Proc Natl Acad Sci U S A 102, no. 23 (2005): 8144-9.

Heath, C. M., M. Windsor, and T. Wileman. "Aggresomes resemble sites specialized for virus assembly." J Cell Biol 153, no. 3 (2001): 449-55.

Once a virus assembles inside a cell, it has to get out. Bacteriophages have a simple but robust system for lysing their hosts, literally causing them to explode. This system has a built-in control mechanism enabling effective timing of lysis. HIV seems to exploit a previously poorly understood cellular pathway for budding from cells. Von Schwedler et al. use genetics to identify over twenty protein players in this very complex pathway.

Grundling, A., D. L. Smith, U. Blasi, and R. Young. "Dimerization between the holin and holin inhibitor of phage lambda." J Bacteriol 182, no. 21 (2000): 6075-81.

von Schwedler, U. K., M. Stuchell, B. Muller, D. M. Ward, H. Y. Chung, E. Morita, H. E. Wang, T. Davis, G. P. He, D. M. Cimbora, A. Scott, H. G. Krausslich, J. Kaplan, S. G. Morham, and W. I. Sundquist. "The protein network of HIV budding." Cell 114, no. 6 (2003): 701-13.

Human Immunodeficiency Virus

Putting together what we have learned about virus structure and assembly, we will discuss HIV antiviral compounds in light of their mechanisms. Viral fusion inhibitors were once a promising anti-HIV therapy. But HIV quickly evolved resistance, and the basis of this resistance is understood in terms of the structure of the protein that mutated, gp41. Sticht et al. report a promising anti-viral peptide that inhibits the assembly of the HIV capsid.

Baldwin, C. E., R. W. Sanders, Y. Deng, S. Jurriaans, J. M. Lange, M. Lu, and B. Berkhout. "Emergence of a drug-dependent human immunodeficiency virus type 1 variant during therapy with the T20 fusion inhibitor." J Virol 78, no. 22 (2004): 12428-37.

Sticht, J., M. Humbert, S. Findlow, J. Bodem, B. Muller, U. Dietrich, J. Werner, and H. G. Krausslich. "A peptide inhibitor of HIV-1 assembly in vitro." Nat Struct Mol Biol 12, no. 8 (2005): 671-7.

Project Day: Looking at Virus Structures in 3D

The visual manipulation of biomacromolecules in 3D is an important tool for structural biologists. We will spend this class period becoming familiar with research-level software that allows us to rotate, manipulate, and view molecules in 3-dimensions, and we will take a molecular-level look at some of the viral proteins discussed during the semester.

Viruses are essentially biological machines. The genetic control of their structures, coupled to the fidelity with which these structures self-assemble, make viruses ideal platforms for the development of nano-scale materials and surfaces.

Mao, C., D. J. Solis, B. D. Reiss, S. T. Kottmann, R. Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson, and A. M. Belcher. "Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires." Science 303, no. 5655 (2004): 213-7.

Meunier, S., E. Strable, and M. G. Finn. "Crosslinking of and coupling to viral capsid proteins by tyrosine oxidation." Chem Biol 11, no. 3 (2004): 319-26.


Methods

Proteins and recombinant DNA

The cloning, over-expression and purification of PP7 coat protein have been described in detail elsewhere. To construct the single-chain PP7 dimer, the coat sequence was amplified from pP7CT with Pfu DNA polymerase and a 3'-primer complementary to plasmid vector sequences and a 5'-primer having the sequence: 5'-CCCCCGCCGTTATGGGCAAAACCATCGTTCTTTCGGTC-3'. This introduced a Bgl I site near the 5'-end of what would be the downstream copy of the coat protein coding sequence. This was subsequently joined to a naturally occurring Bgl I site near the 3'-end of the upstream copy in pP7CT to create the junction sequence shown in Figure 3. The now duplicated sequence was cloned between Xba I and Bam HI in pET3d for over-expression in E. coli. These manipulations resulted in duplication and translational fusion of the two sequences, with the last amino acid of the upstream copy (arginine) joined to the second amino acid (serine) of the downstream copy through a two-amino acid linker (tyr-gly).

Assay for thermal stability

The thermal stabilities of virus-like particles under various conditions were determined by two methods. In the first a "melting profile" was produced by heating 25 ul samples of PP7 virus-like particles at a concentration of 1.0 mg/ml in 50 mM Tris-HCl, pH 8.5, 100 mM NaCl for 2 min. at specific temperatures. When a reaction contained DTT, it was present at a concentration of 10 mM. At the end of the incubation period, the samples were chilled on ice and then and subjected to centrifugation at 13,000 rpm in an IEC MicroMax microcentrifuge for 5 minutes. The supernatants of these samples, containing the portion of the protein that remained soluble after heat treatment, were removed to a new tube. The insoluble proteins in the pellet were redissolved in 6 M urea. Measurements of the relative quantities of soluble and insoluble protein were performed by Bradford assay [24]. Standard curves were produced using hen lysozyme as a standard and were linear over the range of the assay. For measurement of the quantity of capsids remaining after heat treatment, soluble protein was applied to a 1% agarose gel in 40 mM Tris-acetate, pH 8.0, 2 mM EDTA, and subjected to electrophoresis. The gel was then stained with ethidium bromide and photographed under UV illumination to visualize the RNA-containing VLPs. Protein was stained with coomassie brilliant blue R250. The gel was scanned with a densitometer and the quantity of protein in individual bands was determined by comparison to a standard curve produced by applying dilutions of a known quantity of PP7 virus-like particles to the same gel. The standard curve was linear over the range employed in the assay.

The rates of denaturation were determined by incubation of proteins in 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, with or without DTT at 10 mM at specified temperatures. At time points reactions were quenched on ice and then analyzed for their content of capsids and of soluble and insoluble protein as described above.

Purification of dimers

Ten milligrams of PP7 or 2PP7 VLPs purified as described previously were incubated for 60 minutes in 1 ml of 50 mM Tris-HCl, pH 8.5, 6 M urea, 10 mM DTT on ice. The resulting protein was dialyzed against 10 mM acetic acid, 50 mM NaCl (about pH 4) and then applied to a 0.9 × 45 cm column of Sephadex G75 and eluted in the same buffer. Fractions of 0.7 ml were collected. Two peaks appeared in the chromatogram. Agarose gel electrophoresis shows that the first peak is made up of VLPs that failed to disassemble. The other, eluting at fraction 20, apparently represents coat protein dimers. In a separate experiment bovine serum albumin (MW = 68,000), ovalbumin (MW = 45,000), chymotrypsinogen (MW = 25,700) and lysozyme (MW = 14,400) were applied to the column as molecular weight standards. The standard proteins yielded a linear plot of elution position versus log molecular weight. Note that BSA was omitted from this analysis because it eluted in or near the void volume. Comparison to the elution behavior of the standards indicates that the second coat protein peak has a molecular weight of about 32,000, a size roughly consistent with the predicted molecular weight of about 28,000 for the coat protein dimer. Protein from the peak fractions was used in the in vitro assembly reactions.

In vitro VLP assembly

Purified dimeric PP7 coat protein (0.1 nmol) was added to reactions containing 50 mM Tris-HCl, pH 8.5 and yeast tRNA, MS2 translational operator, or PP7 translational operator RNA in amounts varying by two-fold serial dilution from 0.1 nmol to 6.3 pmol. After 30 minutes, glycerol and bromophenol blue were added and the reactions were subjected to electrophoresis in a 1% agarose gel. RNA was visualized by staining the gels with ethidium bromide followed by photography on a UV transilluminator. MS2 and PP7 translational operator RNAs were produced by transcription in vitro as described previously [26]. In some cases RNAs were synthesized in the presence of a 32 P-labeled nucleotide and could be visualized and quantitated after exposure of the gel to a Packard Cyclone phosphorimager screen. To visualize proteins, gels were stained with coomassie brilliant blue R250.


In Vitro Assembly Properties of Purified Bacterially Expressed Capsid Proteins of Human Immunodeficiency Virus

The Gag polyprotein of retroviruses is sufficient for assembly and budding of virus-like particles from the host cell. In the case of human immunodeficiency virus (HIV), Gag contains the domains matrix, capsid (CA), nucleocapsid (NC) and p6 which are separated by the viral proteinase inside the nascent virion, leading to morphological maturation to yield an infectious virus. In the mature virus, CA forms a capsid shell surrounding the ribonucleoprotein core consisting of NC and the genomic RNA. To define requirements for particle assembly and functional contributions of individual domains, we expressed domains of HIV Gag in Escherichia coli and purified the products to near homogeneity. In vitro assembly of CA, with or without the C-terminally adjacent spacer peptide, yielded tubular structures with a diameter of approximately 55 nm and heterogeneous length. Efficient particle formation required high protein concentration, high salt and neutral to alkaline pH. In contrast, in vitro assembly of CA-NC occurred at a 20-fold lower protein concentration and in low salt, but required addition of RNA. These results suggest that hydrophobic interactions of capsid proteins are sufficient for particle formation while the RNA-binding nucleocapsid domain may concentrate and align structural proteins on the viral genome.


Abstract

Synthesis of a small amount of 42S RNA in addition to the VSV specific mRNA species was observed in a coupled transcription-translation system containing ribonucleoprotein particles from L cell infected with vesicular stomatitis virus and nuclease-treated ribosomal extract obtained from uninfected HeLa cells. Analysis on a CsCl density gradient showed that the synthesized 42S RNA was associated with newly synthesized N protein as a nucleoprotein of bouyant density of 1.3 g/ml. The 42S RNA and the N protein present in the nucleoprotein were resistant to nuclease and protease, respectively. About 35% of the synthesized 42S RNA had the same polarity as the VSV genomic RNA and the remaining 65% had a complementary polarity. The evidence presented here demonstrates that both the full length genomic and the complementary RNA are associated with N protein in the in vitro replication process. A template role for the complementary 42S RNA for replication of the genomic RNA is also suggested.


Introduction

The in vitro construction of ribosomes is a topic of rapidly growing interest in systems and synthetic biology. These interests aim to elucidate broad principles that underlie the operation and assembly of the translation apparatus ( Nierhaus, 1990 Erlacher et al, 2011 Polacek, 2011 ), design and build minimal cells to understand origins of life ( Forster and Church, 2006 Jewett and Forster, 2010 ), and enable in vitro evolution to select for ribosomes that have enhanced functions or altered chemical properties ( Cochella and Green, 2004 Wang et al, 2007 Neumann et al, 2010 ). To realize these goals, methods for in vitro ribosome synthesis are needed.

In vitro assembly, or reconstitution, of Escherichia coli ribosomes from purified native ribosomal components into functionally active small (30S) and large (50S) ribosomal subunits was first achieved in pioneering works ∼40 years ago. The conventional 30S subunit reconstitution protocol involves a one-step incubation at 20 mM Mg 2+ and 40°C ( Traub and Nomura, 1968 ), and can be facilitated at lower temperatures by chaperones ( Maki and Culver, 2005 ). The conventional 50S subunit reconstitution protocol involves a non-physiological two-step high-temperature incubation, first at 4 mM Mg 2+ and 44°C, then at 20 mM Mg 2+ and 50°C ( Nierhaus and Dohme, 1974 ).

While studies using the conventional reconstitution approach have revealed many important insights into ribosome assembly ( Nierhaus, 1990 ), inefficiencies in reconstitution make the construction and analysis of engineered variants difficult ( Semrad and Green, 2002 ). For example, conventionally reconstituted 50S subunits made with in vitro-transcribed 23S rRNA (lacking the naturally occurring post-transcriptional modifications) are up to 10 000 times less efficient in reconstitution than those using mature 23S rRNA as measured by the fragment reaction, where single peptide bonds are formed on isolated 50S subunits ( Semrad and Green, 2002 ). Furthermore, the non-physiological two-step conditions for 50S assembly preclude coupling of ribosome synthesis and assembly in a single, integrated system.

In contrast to previous schemes, we aimed to develop an integrated method for the physiological assembly of E. coli ribosomes, in which ribosomes assemble from in vitro-transcribed rRNA and then conduct protein synthesis in the same compartment (Box 1). This approach mimics co-transcription of rRNA and ribosome assembly as it occurs in vivo ( Talkington et al, 2005 Mulder et al, 2010 ). Moreover, it is aligned with a general guiding principle in cell-free synthetic biology namely, that cytoplasmic mimicry provides advantages for reproducing cell-like behavior ( Jewett et al, 2008 Hodgman and Jewett, 2012 ). Here we demonstrate our new method for integrated rRNA synthesis, ribosome assembly, and t ranslation, termed iSAT (Box 1). In addition, we show how iSAT can be utilized to efficiently make a modified ribosome that is highly resistant to the antibiotic clindamycin in a single step. Although Bacillus stearothermophilus ( Green and Noller, 1999 ) and Thermus aquaticus ( Khaitovich et al, 1999 Erlacher et al, 2011 ) have quite active ribosomes reconstituted from in vitro-transcribed 23S rRNA lacking modifications, we focus on E. coli ribosomes because the translation apparatus of E. coli is the best understood and most characterized both biochemically and genetically ( Forster and Church, 2006 Jewett and Forster, 2010 ).


In vitro virus assembly - Biology

Experimental Data Snapshot

  • Method: ELECTRON MICROSCOPY
  • Resolution: 3.30 Å
  • Aggregation State: HELICAL ARRAY 
  • Reconstruction Method: HELICAL 

wwPDB Validation   3D Report Full Report

Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication.

(2019) Proc Natl Acad Sci U S A 116: 4256-4264

  • PubMed: 30787192  Search on PubMedSearch on PubMed Central
  • DOI: 10.1073/pnas.1816417116
  • Primary Citation of Related Structures:  
    6H5Q, 6H5S
  • PubMed Abstract: 

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3 .

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.


Watch the video: Program In Vitro Optimum (October 2022).