Science in China Ser C: Life Sciences © 2007

Science in China Press Springer-Verlag

Structure comparisons of Aedes albopictus densovirus with other parvoviruses CHENG LingPeng1,2, CHEN SenXiong2, Z. H. ZHOU3 & ZHANG JingQiang2† 1

Department of Biomedical Engineering, School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510640, China; 2 State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China; 3 Department of Pathology & Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX77030, USA

Parvoviridae is a family of the smallest viruses known with a wide variety of hosts. The capsid structure of the Aedes albopictus C6/36 cell densovirus (C6/36 DNV) at 1.2-nm resolution was obtained by electron cryomicroscopy (cryoEM) and three-dimensional (3D) image reconstruction. Structure comparisons between the C6/36 DNV and other parvoviruses reveal that the degree of structural similarity between C6/36 DNV and the human parvovirus B19 is higher than that between C6/36 DNV and other insect parvoviruses. The amino acid sequence comparisons of structural and non-structural proteins also reveal higher levels of similarity between C6/36 DNV and parvovirus B19 than those between C6/36 DNV and other parvoviruses. These findings indicate that C6/36 DNV is closely related to the human virus B19, and the former might evolve from the human species other than from other insect viruses. densovirus, three-dimensional structure, amino acid sequence.

Parvoviruses are the smallest viruses known to date, with diameters ranging from 25 to 28 nm. Each mature parvovirus is made up of 2―4 structural proteins (VP1-VP4) encoded by a single open reading frame. Transcriptional regulation and post-transcriptional modification produce proteins with different length at their N termini. The common C termini of the 60 structural proteins make up the icosahedral capsid with triangulation number T = 1. Parvoviruses infect a wide variety of hosts, ranging from arthropods to mammals (including humans). In general, they infect and cause disease in the young. The Parvoviridae family comprises two subfamilies according to their hosts, Parvovirinae that infects mammals and Densovirinae that infects arthropods. Although different cell tropisms of parvoviruses lead to the low homology among different genera, they still share some similar structural features. To date, the crystal structures of several parvoviruses have been determined to atomic or near-atom resolution by X-ray www.scichina.com

www.springerlink.com

crystallography. In addition, the 3D structures of some parvoviruses have been reconstructed at resolutions ranging from 1.0 to 2.0 nm by cryoEM. Example structures include human Parvovirus B19[1,2], Galleria mellonella densovirus (GmDNV)[3], Canine Parvovirus (CPV)[4], Feline Par-vovirus (FPV) [5], Porcine Parvovirus (PPV)[6], Aleutian mink disease parvovirus (ADV)[7], Murine parvovirus (MVM)[8], and Adena-associated virus type 2 (AAV2)[9,10]. We recently reported the 3D structure of C6/36 DNV at 1.4-nm resolution and discussed its structural validity[11]. In this study, we included more virion images by template matching algorithm and refined the structure of the virion to 1.2-nm resolution. We also compared the Received April 25, 2006; accepted June 8, 2006 doi: 10.1007/s11427-007-2036-3 † Corresponding author (email: [email protected]) Supported by the National Natural Science Foundation of China (Grant No. 10274106 to Jingqiang Zhang), Welch Foundation (AU-1492 to Z. H. Zhou) and Natural Science Foundation of Guangdong Province, China (05300232 to Lingpeng Cheng)

Sci China Ser C-Life Sci | February 2007 | vol. 50 | no. 1 | 70-74

3D structure and amino acid sequences of C6/36 DNV with other parvoviruses, revealing that the level of sequence identity between C6/36 DNV and parvovirus B19 is higher than that between C6/36 DNV and other densoviruses.

1 Materials and methods 1.1 Refinement of C6/36 DNV structure and conversion of crystal protein structures Previously[11], we obtained the C6/36 DNV structure at 1.4-nm resolution using IMIRS software package[12], which is based on common-lines algorithm. Using an integrative approach of IMIRS[12] together with the program WINPFT (Hui & Zhou, unpublished), we have refined this structure to 1.2-nm resolution. WINPFT provides an MS Windows graphical user interface integrating utility tools and individual programs ported from the UNIX-based PFT package (kindly provided by Drs. T. Baker and X. Zhang) [13]. WINPFT computes the one-dimensional Polar Fourier Transform (1D PFT) for each virus particle image and compares the 1D PFT with a series of 1D PFTs of 2D projections computed from a preliminary 3D structure model. The projection orientations are distributed evenly within an icosahedral asymmetric unit. The orientation of a particle image is defined as the projection orientation of the projection that most closely matches the particle image based on cross-correlation of the 1D PFTs. The center parameter of each virus particle is refined at the same time. The combined set of particle images from IMIRS and WINPFT orientation determination was then refined and reconstructed by the orientation/center refinement and 3D reconstruction program in IMIRS, respectively. Our experience indicates that integrative use of IMIRS and WINPFT allows us to determine the orientation and

center parameters for more particle images than either program alone. The crystal structures of B19, CPV, and GmDNV were downloaded from Protein Data Bank (PDB). Their PDB ID codes are 1s58, 2cas and 1dnv respectively. Their intact capsid structures were created by icosahedral symmetry operation using program pdb2mrc in the software package EMAN from the National Center for Macromolecular Imaging, USA[14]. The virus structures were then converted to density maps and low-pass filtered to a resolution of 1.2 nm (Figure 1). 1.2 Visualization and fitting of X-ray and cryoEM structures Fitting of the X-ray or NMR structures to cryoEM 3D density maps is widely used to study the viral capsid proteins and other biological macromolecules[15]. All structures were displayed fitted by using the 3D molecular visualization software package Chimera downloaded from the UCSF Computer Graphics Laboratory[16], running in a personal computer with a Pentium 3.0 GHz CPU and 1GM memories. The process of the fitting is to match the two structures through rotation and translation adjustment. 1.3 Sequence alignment The sequence alignment was done using AlignX in the Vector 7.1 software package, which employs Clustal W algorithm[17].

2 Results 2.1 Comparisons of capsid structures among C6/36 DNV and other parvoviruses Due to the increased number of particles used in the 3D reconstruction and the use of integrative methods in determining orientation and center parameters of viral

Figure 1 (a), (b), (c) and (d) are 3D structures of C6/36 DNV, B19, GmDNV and CPV at 1.2-nm resolution viewed along an icosahedral twofold axis. 5, 3, 2 in (a) indicate the fivefold, threefold and twofold icosahedral axes respectively. All the four structures are positioned along the same direction. CHENG LingPeng et al. Sci China Ser C-Life Sci | February 2007 | vol. 50 | no. 1 | 70-74

71

particle images, the C6/36 DNV capsid structure was improved to 1.2-nm resolution, which shows more distinctive structural features than the previous structure at 1.4-nm resolution (Figure 1(a)). Among those parvoviruses whose structures have been determined by X-ray crystallography, we chose three with representative structure features, i.e. B19, CPV and GmDNV, for comparison with our C6/36 DNV. Their capsid structures at 1.2-nm resolution are shown in Figure 1. We can see that these parvoviruses share some prominent structural features. For example, there are depressed regions (or dimples) at the twofold axes of symmetry. Except for the GmDNV, the fivefold positions are dome-shaped, which are surrounded by depressed regions (or canyons), and there are one or three protrusions at or surrounding each threefold axis. In fact, these structural features are conserved among most parvoviruses[18]. It is notable that C6/36 DNV and GmDNV have less common structural features despite that they both infect insect hosts. Likewise, C6/36 DNV has little common structural features with another densovirus, Periplaneta fuliginosa densovirus (pfDNV), whose structural protein has 30.3% sequence homology with that of GmDNV) [19,20]. In contrast, the capsid structures of C6/36 DNV and B19 are quite similar although they have very different hosts. For example, they both have dimples at the twofold and threefold axes of symmetry and spikes at the fivefold axes of symmetry. In addition, the rims surrounding the canyons around the fivefold axes of symmetry at both capsids are extended in a similar way. Superposition of the structures of C6/36 DNV and B19 (Figure 2) shows that the structure of B19 covers the entire surface of C6/36 DNV except the spikes at the fivefold axes of symmetry and nearby regions. Moreover, their inner surfaces match well with each other. These properties are consistent with the fact that the amino acid sequence of the structural protein of B19 is longer than that of C6/36 DNV. 2.2 Sequence comparisons of C6/36 DNV and other parvoviruses Sequence comparisons of main structural proteins reveal that the identities are 11.9%, 11.1% and 11.4% between C6/36 DNV VP1[21] and B19 VP2[1], between C6/36 DNV VP1 and CPV VP2, between C6/36 DNV VP1 and GmDNV VP4, respectively. Similarly, the sequence 72

Figure 2 Superposition of C6/36 DNV and B19. The structure of C6/36 DNV is in red and that of B19 is in gray. The capsid of C6/36 DNV is covered by the capsid of B19 except for the fivefold vertices.

identities between the non-structural protein homologs between these viruses are 12.2%, 10.9% and 10.4% respectively. Therefore, at the amino sequence level, C6/36 DNV is closest to B19 as compared to other parvoviruses. 2.3 Fitting of crystal structure of B19 VP2 and cryoEM structure of C6/36 DNV capsid We fitted the crystal structures of three VP2 molecules of B19 into the C6/36 DNV capsid and found that some segments of B19 VP2 were exposed outside the cryoEM structure while the main portions of two structures fit well with each other (Figure 3(a)). We traced these exposed structures to the sequences marked by black boxes in Figure 4. These exposed sequences correspond to gaps or deletions on the C6/36 DNV VP1 sequence. These exposed sequences include three segments: amino acids 50-P to 68-S attributable to the canyon and the part of rims surrounding the canyon, amino acids 290-P to 327-S attributable to the surfaces beneath the rims and three protrusions surrounding each threefold axis, and amino acids 356-Q to 368-Q attributable to the other part of rims surrounding the canyon. The inner capsid surfaces of B19 and C6/36 DNV match relatively well, except that three β-strands of B19 VP2 parallel to the inner surface of C6/36 DNV capsid somewhat extend the above inner surface (Figure 3(b)). 2.4 Spikes at the fivefold vertices A characteristic structural feature shared by C6/36 DNV and B19 is that there is a cavity beneath each of the 12 spikes at the fivefold vertices, but there is no channel penetrating the capsid in each cavity. Such kind of spike

CHENG LingPeng et al. Sci China Ser C-Life Sci | February 2007 | vol. 50 | no. 1 | 70-74

3 Discussion

Figure 3 (a) Structure of C6/36 DNV viewed along threefold axis (1.2-nm resolution). The crystal structures in red, yellow and blue are 3 VP2 of B19 fitted into an icosahedral surface of C6/36 DNV. (b) Rendering of C6/36 DNV cutting edge (dashed in (a)).

is also found in the same position on CPV capsid, however, there is no cavity underneath the CPV spikes, instead, there is a channel passing through each spike.

Parvoviruses have similar capsid features despite of their diverse cell tropisms and their low level of amino acid identities among their structural proteins. These conserved surface features are known as the receptor binding sites[22], while the similar inner surface structures are consistent with the conserved interaction between the inner surface and the nucleic acid[23]. C6/36 DNV, GmDNV and pfDNV belong to the same subfamily of denovirinae, and share host of insect, but they share few similarities in capsid structure and amino acid sequence. B19 is the only parvovirus that is known to infect human. C6/36 DNV has the capsid structure similar to that of B19, and the levels of amino acid identities between C6/36 DNV and B19 proteins are higher than those between C6/36 DNV and GmDNV or CPV, indicating that C6/36 DNV shares higher homologies with B19. In addition, the close relationship between human and mosquito suggests that C6/36 DNV probably diverges from human parvovirus or other mammalian parvovirus, rather than from insect parvovirus. Compared with structural protein amino acid of B19, there are some sequences that are not found in the amino acid sequence of C6/36 DNV (Figure 4). It is probable that

Figure 4 Alignment of C6/36 DNV VP1 (upper) and B19 VP2 (lower). The amino acids sequences in black boxes correspond to the crystal structures of B19 that cannot be fitted into the cryoEM structure of C6/36 DNV. Aligned identical residues are marked with gray background.

CHENG LingPeng et al. Sci China Ser C-Life Sci | February 2007 | vol. 50 | no. 1 | 70-74

73

these missing sequences are indispensable for parvoviruses to infect human cells, while they are non-essential for parvoviruses to infect mosquito cells. So, these sequences were lost during evolution. The deletion of these sequences might have happened at the early stage of human evolution as judged from the relative low ho1

Kaufmann B, Simpson A A, Rossmann M G. The structure of human

2

Chipman P R, Agbandje-McKenna M, Kajigaya S, et al.

mology level between C6/36 DNV and B19. We thank Dr. Wah Chiu for providing access to the cryoEM facilities at the National Center for Macromolecular Imaging (supported by NIH P41RR02250), Joanita Jakana for assistance in electron cryomicroscopy and Jennifer Brannan for preliminary image processing.

struction package integrated with a relational image database. J Struct

parvovirus B19. Proc Natl Acad Sci USA, 2004, 101: 11628―11633

orientations of biological macromolecules imaged by cryoelectron

virus B19 complexed with cellular receptor. Proc Natl Acad Sci USA,

microscopy. J Struct Biol, 1996, 116: 120―130 14

Simpson A A, Chipman P R, Baker T S, et al. The structure of an inStructure, 1998, 6(11): 1355―1367

5

128(1): 82―97 15

termination of icosahedral viruses. Curr Opin Structl Biol, 2003, 13:

J Mol Biol, 1993, 233(2): 231―244

558―569

Simpson A A, Chandrasekar V, Herbert B, et al. Host range and

16

Chem, 2004, 25: 1605―1612

Simpson A A, Herbert B, Sullivan G M, et al. The structure of porcine

17

sensitivity of progressive multiple sequence alignment through se-

1189―1198

quence weighting, positions-specific gap penalties and weight matrix

McKenna R, Olson N H, Chipman P R, et al. Three-dimensional

choice. Nucleic Acids Res, 1994, 22: 4673―4680 18

Hueffer K, Parrish C R. Parvovirus host range, cell tropism and evo-

19

Li L, Chen D, Zhou Z H, et al. Comparative analysis of the three di-

lution. Curr Opin Microbiol, 2003, 6: 392―398

ease pathogenicity. J Virol, 1999, 73(8): 6882―6891

9

Agbandjie-Mckenna M, Llamas-saiz A L, Wang F, et al. Functional implications of the structure of the murine parvovirus, minute virus of

mensional structure of Periplaneta fuliginosa densovirus. Chin Sci

mice. Structure, 1998, 6: 1369―1381

Bull, 2002, 47(3): 277―281

Kronenberg

S,

Kleinschmidt

J

A,

Bottcher

B.

Electron

20

486―491

type 2 empty capsid. EMBO J, 2001, 2: 997―1002

11

12

Xie Q, Bu W, Bhatia S, et al. The atomic structure of adeno-associated

21

Chen S, Cheng L, Zhang Q, et al. Genetic, biochemical and structural

virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci

characterization of a new densovirus isolated from a chronically in-

USA, 2002, 99: 10405―10410

fected aedes albopicus C6/36 Cell Line. Virology, 2004, 318: 123―133

Cheng L, Chen S, Brannan J M, et al. Three-dimensional structure determination of capsid of Aedes albopicus C6/36 cell densovirus. Sci

22

Colman P M. Virus versus antibody. Structure, 1997, 15(5): 591―593

China Ser C-Life Sci, 2004, 47: 224―228

23

Chapman M S, Rossmann M G. Structure, sequence, and function

Liang Y, Ke E Y, Zhou Z H. IMIRS: a high-resolution 3D recon-

Page 362

74

Li L, Guo H -t, Zhang J-m, et al. Studies on reclassifying of Periplaneta fuliginosa densovirus. Virol Sin (in Chinese), 2003, 18(5):

cryo-microscopy and image reconstruction of adeno-associated virus 10

Thompson J D, Higgins D G, Gibson T J. CLUSTALW: improving the

parvovirus: comparison with related viruses. J Mol Biol, 2002, 315:

structure of Aleutian mink disease parvovirus: implications for dis8

Pettersen E F, Goddard T D, Huang C C, et al. UCSF Chimera - A visualization system for exploratory research and analysis. J Comput

and feline parvoviruses. J Mol Biol, 2000, 300: 597―610

7

Lee K K, Johnson, J E. Complementary approaches to structure de-

Wu H, Rossmann M G. The canine parvovirus empty capsid structure.

variability of calcium binding by surface loops in the capsids of canine 6

Ludtke S J, Baldwin P R, Chiu W. EMAN: Semiautomated software for high-resolution single-particle reconstructions. J Struct Biol, 1999,

sect parvovirus (Galleria mellonella densovirus) at 3.7 Å resolution. 4

Baker T S, Cheng R H. A model-based approach for determining

Cryo-electron microscopy studies of empty capsids of human parvo1996, 93: 7502―7506 3

Biol, 2002, 137(3): 292―304 13

correlations among parvoviruses. Virology, 1993, 194: 491―508

CORRIGENDA Vol. 49 No. 4 2006 Line For 3 ZHU Yinming

Read ZHU Yingmin

CHENG LingPeng et al. Sci China Ser C-Life Sci | February 2007 | vol. 50 | no. 1 | 70-74