Atomic model of vesicular stomatitis virus and mechanism of assembly.

Like other negative-strand RNA viruses (NSVs) such as influenza and rabies, vesicular stomatitis virus (VSV) has a three-layered organization: a layer of matrix protein (M) resides between the glycoprotein (G)-studded membrane envelope and the nucleocapsid, which is composed of the nucleocapsid protein (N) and the encapsidated genomic RNA. Lack of in situ atomic structures of these viral components has limited mechanistic understanding of assembling the bullet-shaped virion. Here, by cryoEM and sub-particle reconstruction, we have determined the in situ structures of M and N inside VSV at 3.47 Å resolution. In the virion, N and M sites have a stoichiometry of 1:2. The in situ structures of both N and M differ from their crystal structures in their N-terminal segments and oligomerization loops. N-RNA, N-N, and N-M-M interactions govern the formation of the capsid. A double layer of M contributes to packaging of the helical nucleocapsid: the inner M (IM) joins neighboring turns of the N helix, while the outer M (OM) contacts G and the membrane envelope. The pseudo-crystalline organization of G is further mapped by cryoET. The mechanism of VSV assembly is delineated by the network interactions of these viral components.

Locations and in situ structure of the polymerase complex inside the virion of vesicular stomatitis virus.

The polymerase complex of nonsegmented negative-strand RNA viruses primarily consists of a large (L) protein and a phosphoprotein (P). L is a multifunctional enzyme carrying out RNA-dependent RNA polymerization and all other steps associated with transcription and replication, while P is the nonenzymatic cofactor, regulating the function and conformation of L. The structure of a purified vesicular stomatitis virus (VSV) polymerase complex containing L and associated P segments has been determined; however, the location and manner of the attachments of L and P within each virion are unknown, limiting our mechanistic understanding of VSV RNA replication and transcription and hindering engineering efforts of this widely used anticancer and vaccine vector. Here, we have used cryo-electron tomography to visualize the VSV virion, revealing the attachment of the ring-shaped L molecules to VSV nucleocapsid proteins (N) throughout the cavity of the bullet-shaped nucleocapsid. Subtomogram averaging and three-dimensional classification of regions containing N and the matrix protein (M) have yielded the in situ structure of the polymerase complex. On average, ∼55 polymerase complexes are packaged in each virion. The capping domain of L interacts with two neighboring N molecules through flexible attachments. P, which exists as a dimer, bridges separate N molecules and the connector and C-terminal domains of L. Our data provide the structural basis for recruitment of L to N by P in virus assembly and for flexible attachments between L and N, which allow a quick response of L in primary transcription upon cell entry.

Overexpression of Smac by an Armed Vesicular Stomatitis Virus Overcomes Tumor Resistance.

Despite reports of successful clinical cases, many tumors appear to resist infection by oncolytic viruses (OVs). To circumvent this problem, an armed vesicular stomatitis virus was constructed by inserting a transgene to express Smac/DIABLO during virus infection (VSV-S). Endogenous Smac in HeLa cells was diminished during wtVSV infection, whereas the Smac level was enhanced during VSV-S infection. Apoptosis was readily induced by VSV-S, but not wtVSV, infection. More importantly, the tumor volume was reduced to a larger extent when xenografts of 4T1 cells in BALB/c mice and OV-resistant T-47D cells in nude mice were intratumorally injected with VSV-S. VSV-S represents a novel mechanism to overcome tumor resistance, resulting in more significant tumor regression due to enhanced apoptosis.

Reduction of Influenza Virus Envelope's Fusogenicity by Viral Fusion Inhibitors.

During cell entry of an enveloped virus, the viral membrane must be fused with the cellular membrane. The virus envelope has a unique structure consisting of viral proteins and a virus-specific lipid composition, whereas the host membrane has its own structure with host membrane proteins. Compound 136 was previously found to bind in close proximity to the viral envelope and inhibit influenza virus entry. We showed here that the 136-treated influenza virus still caused hemolysis. When liposomes were used as the target membrane for 136-treated viruses, aberrant fusion occurred; few liposomes fused per virion, and glycoproteins were not distributed evenly across fusion complexes. Additionally, large fusion aggregates did not form, and in some instances, neck-like structures were found. Based on previous results and hemolysis, fusion inhibition by 136 occurs post-scission but prior to lipid mixing.

Characterization of potent fusion inhibitors of influenza virus.

New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replication of influenza virus at picomolar concentrations in plaque reduction assays. The compound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, inhibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change. This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope required for fusion to cellular membranes.

Structural studies on the authentic mumps virus nucleocapsid showing uncoiling by the phosphoprotein.

Mumps virus (MuV) is a highly contagious pathogen, and despite extensive vaccination campaigns, outbreaks continue to occur worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form the nucleocapsid (NC). NC serves as the template for both transcription and replication. In this paper we solved an 18-Å-resolution structure of the authentic MuV NC using cryo-electron microscopy. We also observed the effects of phosphoprotein (P) binding on the MuV NC structure. The N-terminal domain of P (PNTD) has been shown to bind NC and appeared to induce uncoiling of the helical NC. Additionally, we solved a 25-Å-resolution structure of the authentic MuV NC bound with the C-terminal domain of P (PCTD). The location of the encapsidated viral genomic RNA was defined by modeling crystal structures of homologous negative strand RNA virus Ns in NC. Both the N-terminal and C-terminal domains of MuV P bind NC to participate in access to the genomic RNA by the viral RNA-dependent-RNA polymerase. These results provide critical insights on the structure-function of the MuV NC and the structural alterations that occur through its interactions with P.

Common mechanism for RNA encapsidation by negative-strand RNA viruses.

UNLABELLED: The nucleocapsid of a negative-strand RNA virus is assembled with a single nucleocapsid protein and the viral genomic RNA. The nucleocapsid protein polymerizes along the length of the single-strand genomic RNA (viral RNA) or its cRNA. This process of encapsidation occurs concomitantly with genomic replication. Structural comparisons of several nucleocapsid-like particles show that the mechanism of RNA encapsidation in negative-strand RNA viruses has many common features. Fundamentally, there is a unifying mechanism to keep the capsid protein protomer monomeric prior to encapsidation of viral RNA. In the nucleocapsid, there is a cavity between two globular domains of the nucleocapsid protein where the viral RNA is sequestered. The viral RNA must be transiently released from the nucleocapsid in order to reveal the template RNA sequence for transcription/replication. There are cross-molecular interactions among the protein subunits linearly along the nucleocapsid to stabilize its structure. Empty capsids can form in the absence of RNA. The common characteristics of RNA encapsidation not only delineate the evolutionary relationship of negative-strand RNA viruses but also provide insights into their mechanism of replication. IMPORTANCE: What separates negative-strand RNA viruses (NSVs) from the rest of the virosphere is that the nucleocapsid of NSVs serves as the template for viral RNA synthesis. Their viral RNA-dependent RNA polymerase can induce local conformational changes in the nucleocapsid to temporarily release the RNA genome so that the viral RNA-dependent RNA polymerase can use it as the template for RNA synthesis during both transcription and replication. After RNA synthesis at the local region is completed, the viral RNA-dependent RNA polymerase processes downstream, and the RNA genome is restored in the nucleocapsid. We found that the nucleocapsid assembly of all NSVs shares three essential elements: a monomeric capsid protein protomer, parallel orientation of subunits in the linear nucleocapsid, and a (5H + 3H) motif that forms a proper cavity for sequestration of the RNA. This observation also suggests that all NSVs evolved from a common ancestor that has this unique nucleocapsid.

Posttranscriptional control of type I interferon genes by KSRP in the innate immune response against viral infection.

Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3' untranslated regions. Expression of ARE-containing type I interferon transcripts is robustly induced upon viral infection and rapidly shut off thereafter. Their transient accumulation is partly mediated through posttranscriptional regulation. Here we show that mouse embryonic fibroblasts derived from knockout mice deficient in KH-type splicing regulatory protein (KSRP), an RNA-binding protein required for ARE-mediated mRNA decay, produce higher levels of Ifna and Ifnb mRNAs in response to viral infection as a result of decreased mRNA decay. Functional analysis showed that KSRP is required for the decay of Ifna4 and Ifnb mRNAs by interaction with AREs. The increased IFN expression renders Ksrp(-)(/)(-) cells refractory to herpes simplex virus type 1 and vesicular stomatitis virus infection. These findings support a role of a posttranscriptional mechanism in the control of type I IFN expression and highlight the function of KSRP in innate immunity by negatively regulating IFN production.

Access to RNA encapsidated in the nucleocapsid of vesicular stomatitis virus.

The genomic RNA of negative-strand RNA viruses, such as vesicular stomatitis virus (VSV), is completely enwrapped by the nucleocapsid protein (N) in every stage of virus infection. During viral transcription/replication, however, the genomic RNA in the nucleocapsid must be accessible by the virus-encoded RNA-dependent RNA polymerase in order to serve as the template for RNA synthesis. With the VSV nucleocapsid and a nucleocapsid-like particle (NLP) produced in Escherichia coli, we have found that the RNA in the VSV nucleocapsid can be removed by RNase A, in contrast to what was previously reported. Removal of the RNA did not disrupt the assembly of the N protein, resulting in an empty capsid. Polyribonucleotides were reencapsidated into the empty NLP, and the crystal structures were determined. The crystal structures revealed variable degrees of association of the N protein with a specific RNA sequence.

Cryo-EM model of the bullet-shaped vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Through direct visualization by means of cryo-electron microscopy, we show that each virion contains two nested, left-handed helices: an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid. Side-by-side interactions between neighboring N subunits are critical for the nucleocapsid to form a bullet shape, and structure-based mutagenesis results support this description. Together, our data suggest a mechanism of VSV assembly in which the nucleocapsid spirals from the tip to become the helical trunk, both subsequently framed and rigidified by the M layer.

Characterization of a mumps virus nucleocapsidlike particle.

The nucleocapsid protein (NP) of mumps virus (MuV), a paramyxovirus, was coexpressed with the phosphoprotein (P) in Escherichia coli. The NP and P proteins form a soluble complex containing RNA. Under a transmission electron microscope, the NP-RNA complex appears as a nucleocapsidlike ring that has a diameter of approximately 20 nm with 13 subunits. There is a piece of single-stranded RNA with a length of 78 nucleotides in the NP-RNA ring. Shorter RNA pieces are also visible. The MuV NP protein may provide weaker protection of the RNA than the NP protein of some other negative-strand RNA viruses, reflecting the degree of NP protein association.

Role of intermolecular interactions of vesicular stomatitis virus nucleoprotein in RNA encapsidation.

The crystal structure of the vesicular stomatitis virus nucleoprotein (N) in complex with RNA reveals extensive and specific intermolecular interactions among the N molecules in the 10-member oligomer. What roles these interactions play in encapsidating RNA was studied by mutagenesis of the N protein. Three N mutants intended for disruption of the intermolecular interactions were designed and coexpressed with the phosphoprotein (P) in an Escherichia coli system previously described (T. J. Green et al., J. Virol. 74:9515-9524, 2000). Mutants N (Delta1-22), N (Delta347-352), and N (320-324, (Ala)(5)) lost RNA encapsidation and oligomerization but still bound with P. Another mutant, N (Ser290-->Trp), was able to form a stable ring-like N oligomer and bind with the P protein but was no longer able to encapsidate RNA. The crystal structure of N (Ser290-->Trp) at 2.8 A resolution showed that this mutant can maintain all the same intermolecular interactions as the wild-type N except for a slight unwinding of the N-terminal lobe. These results suggest that the intermolecular contacts among the N molecules are required for encapsidation of the viral RNA.

Conserved characteristics of the rhabdovirus nucleoprotein.

Rhabdovirus is a negative strand RNA virus that packages a ribonucleoprotein (RNP) complex. The RNP is composed of a genome that is encapsidated completely by the nucleoprotein (N). Structural comparisons of the RNA-nucleoprotein complexes from two members, vesicular stomatitis virus (VSV) and rabies virus (RABV), revealed highly conserved characteristics of folding, RNA binding, and assembly despite their lack of significant homology in amino acid sequence. The RNA binding cavity is located between two conserved domains formed by alpha-helices, but the positively charged residues that coordinate with the phosphate groups are at different sites. The intermolecular interactions among N molecules have a conserved pattern that is rendered, however, by different residues. The curvature of the RABV N-RNA complex in the crystal structure is larger than that of the VSV N-RNA complex. The more relaxed curvature allows the bases in the RNA to stack more tightly, and at the same time, the helices near the C-terminus move into the created space in order to cover the bound RNA. This may explain how the RNP can adopt different conformations from being packed as a superhelix in the virion to a relaxed linear structure once being delivered into the cytoplasm.

Structural comparisons of the nucleoprotein from three negative strand RNA virus families.

Structures of the nucleoprotein of three negative strand RNA virus families, borna disease virus, rhabdovirus and influenza A virus, are now available. Structural comparisons showed that the topology of the RNA binding region from the three proteins is very similar. The RNA was shown to fit into a cavity formed by the two distinct domains of the RNA binding region in the rhabdovirus nucleoprotein. Two helices connecting the two domains characterize the center of the cavity. The nucleoproteins contain at least 5 conserved helices in the N-terminal domain and 3 conserved helices in the C-terminal domain. Since all negative strand RNA viruses are required to have the ribonucleoprotein complex as their active genomic templates, it is perceivable that the (5H+3H) structure is a common motif in the nucleoprotein of negative strand RNA viruses.

SGCEdb: a flexible database and web interface integrating experimental results and analysis for structural genomics focusing on Caenorhabditis elegans.

The SGCEdb (http://sgce.cbse.uab.edu) database/interface serves the primary purpose of reporting progress of the Structural Genomics of Caenorhabditis elegans project at the University of Alabama at Birmingham. It stores and analyzes results of experiments ranging from solubility screening arrays to individual protein purification and structure solution. External databases and algorithms are referenced and evaluated for target selection in the human, C.elegans and Pneumocystis carinii genomes. The flexible and reusable design permits tracking of standard and custom experiment types in a scientist-defined sequence. The database coordinates efforts between collaborators and is adaptable to a wide range of biological applications.

High-throughput expression of C. elegans proteins.

Proteome-scale studies of protein three-dimensional structures should provide valuable information for both investigating basic biology and developing therapeutics. Critical for these endeavors is the expression of recombinant proteins. We selected Caenorhabditis elegans as our model organism in a structural proteomics initiative because of the high quality of its genome sequence and the availability of its ORFeome, protein-encoding open reading frames (ORFs), in a flexible recombinational cloning format. We developed a robotic pipeline for recombinant protein expression, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. Using the pipeline, we have carried out heterologous protein expression experiments on 10,167 ORFs of C. elegans. With one expression vector and one Escherichia coli strain, protein expression was observed for 4854 ORFs, and 1536 were soluble. Bioinformatics analysis of the data indicates that protein hydrophobicity is a key determining factor for an ORF to yield a soluble expression product. This protein expression effort has investigated the largest number of genes in any organism to date. The pipeline described here is applicable to high-throughput expression of recombinant proteins for other species, both prokaryotic and eukaryotic, provided that ORFeome resources become available.

Crystal structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain.

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.