The three-dimensional atomic structure of a single-stranded DNA virus has been determined. Infectious virions of canine parvovirus contain 60 protein subunits that are predominantly VP-2. The central structural motif of VP-2 has the same topology (an eight-stranded antiparallel beta barrel) as has been found in many other icosahedral viruses but represents only about one-third of the capsid protein. There is a 22 angstrom (A) long protrusion on the threefold axes, a 15 A deep canyon circulating about each of the five cylindrical structures at the fivefold axes, and a 15 A deep depression at the twofold axes. By analogy with rhinoviruses, the canyon may be the site of receptor attachment. Residues related to the antigenic properties of the virus are found on the threefold protrusions. Some of the amino termini of VP-2 run to the exterior in full but not empty virions, which is consistent with the observation that some VP-2 polypeptides in full particles can be cleaved by trypsin. Eleven nucleotides are seen in each of 60 symmetry-related pockets on the interior surface of the capsid and together account for 13 percent of the genome.

Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Based on direct visualization by 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.

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 (P NTD ) 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 (P CTD ). 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 structurefunction of the MuV NC and the structural alterations that occur through its interactions with P.replication | paramyxovirus | mononegavirale P aramyxoviruses are enveloped nonsegmented negative-strand RNA viruses (NSV) belonging to the order Mononegavirales. Mononegavirales also includes the Bornaviridae, Filoviridae, and Rhabdoviridae families. The Paramyxoviridae family includes several important human pathogens such as measles virus (MeV), respiratory syncytial virus (RSV), and mumps virus (MuV). Although vaccines exist for some paramyxoviruses, they are not available for others, such as RSV. In addition, no effective antiviral treatments have been developed.The MuV genome encodes 9 proteins, three of which are required for replication of the MuV genome; the nucleocapsid protein (N), phosphoprotein (P), and the large protein (L). N, P, and L have orthologs in a number of NSV. Studies on the roles of N, P, and L in viral RNA synthesis have shown that each can individually and differentially affect the processes of mRNA transcription and genome replication (1-10).Throughout the virus replication cycle, the genome of NSV always exists in the nucleocapsid (NC), a unique protein-RNA complex in which the viral RNA [viral genomic RNA (vRNA) or complementary genomic RNA (cRNA)] is completely sequestered by the N protein. NC is used as the functional template for RNA synthesis by the viral RNA dependent RNA polymerase (vRdRp), which includes L and P. The L protein contains all of the enzymatic activities needed for viral RNA synthesis, such as the ability to cap and polyadenylate mRNA transcripts. P acts as a cofactor to home vRdRp onto the NC template for RNA synthesis. In addition, the P protein chaperones monomeric and RNA-free N to encapsidate newly synthesized viral genomes during replication. The encapsidation of RNA by N is concomitant with the replication process.How the sequestered vRNA is accessed by vRdRp ...

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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 (⌬1-22), N (⌬347-352), and N (320-324, (Ala) 5 ) lost RNA encapsidation and oligomerization but still bound with P. Another mutant, N (Ser2903Trp), 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 (Ser2903Trp) at 2.8 Å 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.All negative-strand RNA viruses contain a ribonucleoprotein (RNP) complex that consists of the viral genome RNA completely enwrapped by the nucleoprotein (N). Vesicular stomatitis virus (VSV) is a nonsegmented negative-strand RNA virus that belongs to the rhabdovirus family. The genome of VSV encodes five proteins: the nucleoprotein (N), the phosphoprotien (P), the matrix protein (M), the glycoprotein (G), and the large subunit of the polymerase (L). The RNP of VSV is packaged in a bullet-shaped virion by M, the protein that condenses the RNP during virion assembly, and G, the surface protein that is embedded in the viral envelope (14,16,22,29). P and L, which are other viral proteins, are packaged through their association with the RNP. After entry into the cytoplasm via membrane fusion mediated by G, the RNP is released from the virion and serves as the active template with which the copackaged polymerase proteins transcribe mRNAs from the five viral genes in the RNP. In the later stage of the virus replication cycle, a positive strand of the viral genome (cRNA) is produced in the form of an RNP. The cRNA-containing RNP then serves as the template for replication that also generates the viral genomic RNA in the form of an RNP ready to be packaged in the virion. Throughout the entire virus replication cycle of a negative-strand RNA virus, the genomelength viral RNA (cRNA or viral genomic RNA) is only present in the form of an RNP that is either serving as a template for RNA synthesis or being packaged in the virion. The assembly of the RNP is therefore a critical step in the replication of negative-strand RNA viruses.Functions of the N protein require it to have two essential properties. First, the N protein needs to bind a single-strand RNA. Second, the N protein has to polymerize in order to cover the entire length of the viral genome RNA. A number of experiments have attempted to...

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus of the Cardiovirus genus. Certain strains of TMEV may cause a chronic demyelinating disease, which is very similar to multiple sclerosis in humans, associated with a persistent viral infection in the mouse central nervous system (CNS). Other strains of TMEV only cause an acute infection without persistence in the CNS. It has been shown that sialic acid is a receptor moiety only for the persistent TMEV strains and not for the nonpersistent strains. We report the effect of sialylation on cell surface on entry and the complex structure of DA virus, a persistent TMEV, and the receptor moiety mimic, sialyllactose, refined to a resolution of 3.0 Å. The ligand binds to a pocket on the viral surface, composed mainly of the amino acid residues from capsid protein VP2 puff B, in the vicinity of the VP1 loop and VP3 C terminus. The interaction of the receptor moiety with the persistent DA strain provides new understanding for the demyelinating persistent infection in the mouse CNS by TMEV.Theiler's murine encephalomyelitis virus (TMEV) belongs to the Cardiovirus genus of the family Picornaviridae based on sequence analysis (34). Based on the characteristics of the disease they cause, TMEV strains are divided into two groups. Viruses of one group, including strain GDVII and FA, are highly neurovirulent, causing a rapid destruction of the neuron and killing their host in a matter of days. These viruses are unable to persist and to induce demyelination in those rare survivors (22). The other group, including strains DA, BeAn, WW, TO4, and Yale, are referred to as the Theiler's original (TO) group (23). Viruses of the TO group are much less virulent than GDVII and FA and cause a biphasic disease with mild central nervous system (CNS) damage. The first phase, or early onset, is acute encephalitis that occurs during the initial few days following intracranial inoculation. At that time, the virus is also found in neurons of the gray matter in the brain and spinal cord. However, the number of infected cells is small and most of the animals survive. Soon after, the neurons are cleared of the virus and the disease enters a second phase, or late onset, during which the virus is found to be persistent in the white matter of the spinal cord. At this stage of the viral persistence, patchy inflammation and demyelination develops in the spinal cord at the sites of infection (7, 23). The symptoms of the demyelination disease of mice caused by TMEV persistence infection in the CNS resemble those of multiple sclerosis (MS), a chronic demyelinating disease of the human CNS. Among the animal models of virus-induced demyelination in the last two decades, TMEV infection has emerged as one of the best models for studying MS.Although these two groups of TMEV strains have both been shown to replicate well in BHK-21 cells and share high amino acid sequence homology (31, 34-36), they apparently display different pathogenesis characteristics in vivo. Many efforts have been taken to map ...

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.