Atomic model of CPV reveals the mechanism used by this single-shelled virus to economically carry out functions conserved in multishelled reoviruses

Unlike the multishelled viruses in the Reoviridae, cytoplasmic polyhedrosis virus (CPV) is single shelled, yet stable and fully capable of carrying out functions conserved within Reoviridae. Here, we report a 3.1 ? resolution cryo electron microscopy structure of CPV and derive its atomic model, consisting of 60 turret proteins (TPs), 120 each of capsid shell proteins (CSPs) and large protrusion proteins (LPPs). Two unique segments of CSP contribute to CPV's stability: an inserted protrusion domain interacting with neighboring proteins, and an N-anchor tying up CSPs together through strong interactions such as β sheet augmentation. Without the need to interact with outer shell proteins, LPP retains only the N-terminal two-third region containing a conserved helix-barrel core and interacts exclusively with CSP. TP is also simplified, containing only domains involved in RNA capping. Our results illustrate how CPV proteins have evolved in a coordinative manner to economically carry out their conserved functions.     

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.     

Structures of host range-controlling regions of the capsids of canine and feline parvoviruses and mutants

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) differ in their ability to infect dogs and dog cells. Canine cell infection is a specific property of CPV and depends on the ability of the virus to bind the canine transferrin receptor (TfR), as well as other unidentified factors. Three regions in the capsid structure, located around VP2 residues 93, 300, and 323, can all influence canine TfR binding and canine cell infection. These regions were compared in the CPV and FPV capsid structures that have been determined, as well as in two new structures of CPV capsids that contain substitutions of the VP2 Asn-93 to Asp and Arg, respectively. The new structures, determined by X-ray crystallography to 3.2 and 3.3 A resolutions, respectively, clearly showed differences in the interactions of residue 93 with an adjacent loop on the capsid surface. Each of the three regions show small differences in structure, but each appears to be structurally independent of the others, and the changes likely act together to affect the ability of the capsid to bind the canine TfR and to infect canine cells. This emphasizes the complex nature of capsid alterations that change the virus-cell interaction to allow infection of cells from different hosts.     

家蚕质多角体病毒（BmCPV）结构研究

用负染和冷冻电镜技术以及计算机数据处理方法,研究了ＣＰＶ２和空病毒的结构,完整病毒和空病毒的结构和生化组成比较表明,ＣＰＶ具有单层衣壳结构,病毒的５种结构蛋白都位于该单层衣壳上。该单层衣壳按Ｔ＝１的对称结构排列,在二十面体的顶点具有塔状突起。空病毒与完整病毒具有相同的衣壳结构,但内部结构却不相同。     

The structure of a cypovirus and the functional organization of dsRNA viruses.

Cytoplasmic polyhedrosis virus (CPV) is unique among the double-stranded RNA viruses of the family Reoviridae in having a single capsid layer. Analysis by cryo-electron microscopy allows comparison of the single shelled CPV and orthoreovirus with the high resolution crystal structure of the inner shell of the bluetongue virus (BTV) core. This suggests that the novel arrangement identified in BTV, of 120 protein subunits in a so-called 'T=2' organization, is a characteristic of the Reoviridae and allows us to delineate structural similarities and differences between two subgroups of the family--the turreted and the smooth-core viruses. This in turn suggests a coherent picture of the structural organization of many dsRNA viruses.     

昆虫质型多角体病毒的研究进展

质型多角体病毒(Cytoplasmic polyhedrosis virus,CPV)隶属呼肠孤病毒科Reoviridae质型多角体病毒属Cypovirus,通常基因组由10个节段双链RNA构成。RNA分子量为3～27u。根据病毒基因组dsRNA片段在聚丙烯酰胺或琼脂糖凝胶中电泳图谱的差异,目前CPV已被分为19个电泳型。不同于呼肠孤病毒科其它成员,CPV为单层衣壳,而不是常见的双层衣壳结构,衣壳蛋白主要由衣壳蛋白、大突起蛋白及塔式突起蛋白组成。大部分质型多角体病毒引起昆虫慢性疾病,造成寄主死亡或适应性降低。随着RNA病毒基因序列测定技术的成熟,质型多角体病毒的序列测定方面取得较大进展,目前GenBank核苷酸序列数据库中已经公布了家蚕Bombyx mori CPV电泳型1两个株系(H株和I株)、舞毒蛾Lymantria dispar CPV电泳型1、舞毒蛾CPV电泳型14及粉纹夜蛾Trichoplusia ni CPV电泳型全基因组序列,为该病毒的进化与起源的研究提供更多的遗传信息。本文从结构功能、侵染特点、基因组特点及应用前景等方面综述了昆虫质型多角体病毒的研究进展。     

The emergence and evolution of canine parvovirus—an example of recent host range mutation

Canine parvovirus (CPV) emerged in 1978 an a new pathogen of dogs, which spread around the world and now appears endemic in the domesticated and wild dog populations in all countries. CPV is over 98% identical in DNA sequence to viruses which had been known for many years in cats, mink and raccoons, and genetic analysis has revealed that the differences in canine host range are determined by a small number of changes in the capsid protein gene. Comparison of the atomic structures of the CPV and FPV capsids shows that the changes affecting host range and virus-specific antigenic sites are exposed on the capsid surface in three different positions within a raised region at the threefold axis of symmetry, which is also the site of major antigenic determinants on the capsid. Three types of CPV have been defined by antigenic analysis with monoclonal antibodies. The original CPV strain (called CPV type-2) was only present in nature for a few years, and by 1981 it had been largely replaced in nature by a variant of CPV (CPV type 2a), which in turn replaced between 1984 and 1990 by a further variant (CPV type-2b). Those viruses differed by less than 0.2% of their genome sequences, but in each case the replacement apparently occurred on a global scale. The true ancestry of CPV is not clear, but the apparent emergence of the new types of CPV and its subsequent evolution suggest that this is a useful model for the emergence of new viruses with extended host ranges and their continuing adaptation.     

Structure of adeno-associated virus serotype 5

Adeno-associated virus serotype 5 (AAV5) requires sialic acid on host cells to bind and infect. Other parvoviruses, including Aleutian mink disease parvovirus (ADV), canine parvovirus (CPV), minute virus of mice, and bovine parvovirus, also bind sialic acid. Hence, structural homology may explain this functional homology. The amino acids required for CPV sialic acid binding map to a site at the icosahedral twofold axes of the capsid. In contrast to AAV5, AAV2 does not bind sialic acid, but rather binds heparan sulfate proteoglycans at its threefold axes of symmetry. To explore the structure-function relationships among parvoviruses with respect to cell receptor attachment, we determined the structure of AAV5 by cryo-electron microscopy (cryo-EM) and image reconstruction at a resolution of 16 A. Surface features common to some parvoviruses, namely depressions encircling the fivefold axes and protrusions at or surrounding the threefold axes, are preserved in the AAV5 capsid. However, even though there were some similarities, a comparison of the AAV5 structure with those of ADV and CPV failed to reveal a feature which could account for the sialic acid binding phenotype common to all three viruses. In contrast, the overall surface topologies of AAV5 and AAV2 are similar. A pseudo-atomic model generated for AAV5 based on the crystal structure of AAV2 and constrained by the AAV5 cryo-EM envelope revealed differences only in surface loop regions. Surprisingly, the surface topologies of AAV5 and AAV2 are remarkably similar to that of ADV despite only exhibiting approximately 20% identity in amino acid sequences. Thus, capsid surface features are shared among parvoviruses and may not be unique to their replication phenotypes, i.e., whether they require a helper or are autonomous. Furthermore, specific surface features alone do not explain the variability in carbohydrate requirements for host cell receptor interactions among parvoviruses.     

Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus.

Analysis of canine parvovirus (CPV) isolates with a panel of monoclonal antibodies showed that after 1986, most viruses isolated from dogs in many parts of the United States differed antigenically from the viruses isolated prior to that date. The new antigenic type (designated CPV type 2b) has largely replaced the previous antigenic type (CPV type 2a) among virus isolates from the United States. This represents the second occurrence of a new antigenic type of this DNA virus since its emergence in 1978, as the original CPV type (CPV type 2) had previously been replaced between 1979 and 1981 by the CPV type 2a strain. DNA sequence comparisons showed that CPV types 2b and 2a differed by as few as two nonsynonymous (amino acid-changing) nucleotide substitutions in the VP-1 and VP-2 capsid protein genes. One mutation, resulting in an Asn-Asp difference at residue 426 in the VP-2 sequence, was shown by comparison with a neutralization-escape mutant selected with a non-CPV type 2b-reactive monoclonal antibody to determine the antigenic change. The mutation selected by that monoclonal antibody, a His-Tyr difference in VP-2 amino acid 222, was immediately adjacent to residue 426 in the three-dimensional structure of the CPV capsid. The CPV type 2b isolates are phylogenetically closely related to the CPV type 2a isolates and are probably derived from a common ancestor. Phylogenetic analysis showed a progressive evolution away from the original CPV type. This pattern of viral evolution appears most similar to that seen in some influenza A viruses.     

Three-Dimensional Structure of Aleutian Mink Disease Parvovirus: Implications for Disease Pathogenicity

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.     

Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid.

We analyzed a region of the capsid of canine parvovirus (CPV) which determines the ability of the virus to infect canine cells. This region is distinct from those previously shown to determine the canine host range differences between CPV and feline panleukopenia virus. It lies on a ridge of the threefold spike of the capsid and is comprised of five interacting loops from three capsid protein monomers. We analyzed 12 mutants of CPV which contained amino acid changes in two adjacent loops exposed on the surface of this region. Nine mutants infected and grew in feline cells but were restricted in replication in one or the other of two canine cell lines tested. Three other mutants whose genomes contain mutations which affect one probable interchain bond were nonviable and could not be propagated in either canine or feline cells, although the VP1 and VP2 proteins from those mutants produced empty capsids when expressed from a plasmid vector. Although wild-type and mutant capsids bound to canine and feline cells in similar amounts, infection or viral DNA replication was greatly reduced after inoculation of canine cells with most of the mutants. The viral genomes of two host range-restricted mutants and two nonviable mutants replicated to wild-type levels in both feline and canine cells upon transfection with plasmid clones. The capsids of wild-type CPV and two mutants were similar in susceptibility to heat inactivation, but one of those mutants and one other were more stable against urea denaturation. Most mutations in this structural region altered the ability of monoclonal antibodies to recognize epitopes within a major neutralizing antigenic site, and that site could be subdivided into a number of distinct epitopes. These results argue that a specific structure of this region is required for CPV to retain its canine host range.     

The VP1 N-terminal sequence of canine parvovirus affects nuclear transport of capsids and efficient cell infection.

The unique N-terminal region of the parvovirus VP1 capsid protein is required for infectivity by the capsids but is not required for capsid assembly. The VP1 N terminus contains a number of groups of basic amino acids which resemble classical nuclear localization sequences, including a conserved sequence near the N terminus comprised of four basic amino acids, which in a peptide can act to transport other proteins into the cell nucleus. Testing with a monoclonal antibody recognizing residues 2 to 13 of VP1 (anti-VP1-2-13) and with a rabbit polyclonal serum against the entire VP1 unique region showed that the VP1 unique region was not exposed on purified capsids but that it became exposed after treatment of the capsids with heat (55 to 75 degrees C), or urea (3 to 5 M). A high concentration of anti-VP1-2-13 neutralized canine parvovirus (CPV) when it was incubated with the virus prior to inoculation of cells. Both antibodies blocked infection when injected into cells prior to virus inoculation, but neither prevented infection by coinjected infectious plasmid DNA. The VP1 unique region could be detected 4 and 8 h after the virus capsids were injected into cells, and that sequence exposure appeared to be correlated with nuclear transport of the capsids. To examine the role of the VP1 N terminus in infection, we altered that sequence in CPV, and some of those changes made the capsids inefficient at cell infection.     

The structure of porcine parvovirus: comparison with related viruses

The structure of baculovirus-expressed porcine parvovirus (PPV) capsids was solved using X-ray crystallography and was found to be similar to the related canine parvovirus (CPV) and minute virus of mice (MVM). The PPV capsid protein has 57 % and 49 % amino acid sequence identity with CPV and MVM, respectively, but the degree of conservation of surface-exposed residues is lower than average. Consequently, most of the structural differences are on the surface and are the probable cause of the known variability in antigenicity and host range. The NADL-2 and Kresse strains of PPV have distinct tissue tropisms and pathogenicity, which are mediated by one or more of the amino acid residues 381, 386, and 436. These residues are on or near the surface of the virus capsid, where they are likely to be associated with virus-cell interactions.     

Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells

A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.     

The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor

Canine parvovirus (CPV) is a host range variant of a feline virus that acquired the ability to infect dogs through changes in its capsid protein. Canine and feline viruses both use the feline transferrin receptor (TfR) to infect feline cells, and here we show that CPV infects canine cells through its ability to specifically bind the canine TfR. Receptor binding on host cells at 37 degrees C only partially correlated with the host ranges of the viruses, and an intermediate virus strain (CPV type 2) bound to higher levels on cells than did either the feline panleukopenia virus or a later strain of CPV. During the process of adaptation to dogs the later variant strain of CPV gained the ability to more efficiently use the canine TfR for infection and also showed reduced binding to feline and canine cells compared to CPV type 2. Differences on the top and the side of the threefold spike of the capsid surface controlled specific TfR binding and the efficiency of binding to feline and canine cells, and these differences also determined the cell infection properties of the viruses.     

Expression, purification, and characterization of VP2 capsid protein of canine parvovirus in Escherichia coli

The capsid protein VP2 of canine parvovirus (CPV) was expressed in Escherichia coli using pMAL-c2x vector. After codon optimization, the mutant resulted in high-level expression of fusion protein. A simple procedure was used to purify fusion protein and remove endotoxin from fusion protein preparations. The fusion protein was antigenical similar to the native capsid protein as Western-blot assay performed with polyclonal antibodies obtained from dogs vaccinated with CPV. The immunogenicity of fusion protein was demonstrated in a vaccination experiment conducted with rabbits subcutaneously immunized. Rabbits vaccinated with fusion protein developed high titers of virus-specific antibodies response that neutralized CPV in vitro.     

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.     

Structural evolution of reoviridae revealed by oryzavirus in acquiring the second capsid shell

The conservation of the core structure and diversification of the external features among the turreted reoviruses appear to be relevant to structural evolution in facilitating the infection of diverse host species. The structure of Rice ragged stunt virus (RRSV), in the genus Oryzavirus of the family Reoviridae, is determined to show a core composed of capsid shell, clamps, and long turrets. The RRSV core structure is equivalent to the core structure of Orthoreovirus and the virion structure of Cytoplasmic polyhedrosis virus (CPV). In RRSV, five peripheral trimers surround each long turret and sit at the Q trimer position in the T=13l icosahedral symmetry, a structural feature unique to turreted reoviruses. That is, the core of RRSV is partially covered by 60 copies of the peripheral trimer. In contrast, the core of Orthoreovirus is covered by 200 copies of the trimer that sit at the Q, R, S, and T trimer positions. Our results suggest that among the three viruses, RRSV has a structure intermediate between that of Orthoreovirus and the CPV virion. This conclusion coincides with the results of the phylogenetic analysis of amino acid sequences of RNA-dependent RNA polymerases.