The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm(3). The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.

In polyomaviruses the pentameric capsomers are interlinked by the long C-terminal arm of the structural protein VP1. The T‫7؍‬ icosahedral structure of these viruses is possible due to an intriguing adaptability of this linker arm to the different local environments in the capsid. To explore the assembly process, we have compared the structure of two virus-like particles (VLPs) formed, as we found, in a calcium-dependent manner by the VP1 protein of human polyomavirus BK. The structures were determined using electron cryomicroscopy (cryo-EM), and the three-dimensional reconstructions were interpreted by atomic modeling. In the small VP1 particle, 26.4 nm in diameter, the pentameric capsomers form an icosahedral T‫1؍‬ surface lattice with meeting densities at the threefold axes that interlinked three capsomers. In the larger particle, 50.6 nm in diameter, the capsomers form a T‫7؍‬ icosahedral shell with three unique contacts. A folding model of the BKV VP1 protein was obtained by alignment with the VP1 protein of simian virus 40 (SV40). The model fitted well into the cryo-EM density of the T‫7؍‬ particle. However, residues 297 to 362 of the C-terminal arm had to be remodeled to accommodate the higher curvature of the T‫1؍‬ particle. The loops, before and after the C-terminal short helix, were shown to provide the hinges that allowed curvature variation in the particle shell. The meeting densities seen at the threefold axes in the T‫1؍‬ particle were consistent with the triple-helix interlinking contact at the local threefold axes in the T‫7؍‬ structure.The BK virus (BKV) is a human virus belonging to the Polyomaviridae family. It is a nonenveloped virus (ϳ50.0 nm in diameter) with a circular double-stranded DNA genome (ϳ5 kb). The capsid has icosahedral symmetry and is built of 72 capsomers that are all pentamers of the protein VP1 arranged in a Tϭ7 icosahedral lattice (21). All known polyomaviruses have three structural proteins (VP1, VP2, and VP3), of which VP1 is the major capsid protein. Overall amino acid sequence homology between BKV and the other human polyomavirus, JCV, is 75%, and that with the simian polyomavirus (SV40) is 69% (9). In the VP1 protein the sequence similarity rises to 77% and 74% for the JCV and SV40, respectively (35). Due to the high similarity, the solved atomic structure of VP1 of SV40 provides us a template to create a model of the BKV VP1 protein folding.The structures of the SV40 and murine polyomavirus have been determined and show similar features to that seen in the BKV (1, 12). The VP1 pentamer of SV40 and murine polyomavirus is built as a ring of five ␤-barrel-shaped VP1 monomers, tightly linked by interacting loops between the framework of ␤-strands (22,33,34,40). The C-terminal subdomain of each VP1 monomer "invades" a neighboring pentamer, thereby tying the pentamers together in the virion shell. There are six unique monomers building up the capsid (monomer ␣, ␣Ј, and ␣Љ at the local threefold; ␤, ␤Ј around the icosahedral threefold, and ␥ at the twofold) (34). The major s...

bWe established a reverse genetics system for Nyamanini virus (NYMV) and recovered green fluorescent protein (GFP)-expressing virus from full-length cDNA. Using this technology, we assessed the functions of two poorly characterized viral genes. NYMV lacking open reading frame 2 (ORF2) could not be rescued, whereas virus lacking ORF4 was replication competent. ORF4-deficient NYMV readily established a persisting noncytolytic infection but failed to produce infectious viral particles, supporting the view that ORF4 represents an essential factor for NYMV particle assembly. N yamanini virus (NYMV) is the prototype member of a novel genus in the order Mononegavirales, designated "Nyavirus" (1). Recently, Kuhn and coworkers (2) proposed classifying NYMV as a member of a new mononegaviral family, designated "Nyamiviridae." NYMV replicates in the nucleus of infected cells (3), and viral budding was proposed to occur at the plasma membrane (1). The NYMV genome contains six major open reading frames (ORFs). The viral nucleoprotein N, the glycoprotein G, and the polymerase L are encoded by ORF1, ORF5, and ORF6, respectively. We recently showed that ORF3 codes for a polymerase cofactor which is required for NYMV polymerase activity (3). The functions of the proteins encoded by ORF2 and ORF4 remain largely unknown. ORF2 negatively regulates NYMV polymerase activity, presumably through interaction with the ORF3 product (3). ORF2 was further shown to promote production of infectious virus-like particles by an unknown mechanism (3). Formation of virus-like particles required the simultaneous presence of the viral G protein, the product of ORF4, and the product of ORF2, indicating that ORF2 and ORF4 both play a role in NYMV particle assembly (3). It remains unclear, however, whether ORF4 represents the NYMV-specific equivalent of a matrix protein typically found in negative-strand RNA viruses. No substantial homology was detected between ORF4 of NYMV and matrix proteins of other members of the order Mononegavirales (1).Matrix (M) proteins of negative-strand RNA viruses orchestrate the assembly of viral particles by binding to both the viral ribonucleoprotein (RNP) complex and cellular membranes (reviewed in references 4 and 5). The importance of M proteins for the assembly and budding of negative-strand RNA viruses was directly demonstrated for rabies virus, measles virus, and Sendai virus by employing reverse genetics approaches (6-8). M-deficient variants of these viruses showed extensive cell-to-cell spreading but failed to produce cell-free infectious particles.To further characterize the ORF2 and ORF4 genes of NYMV, we set out to establish a reverse genetics system for recovering recombinant virus from cloned cDNA. Using cDNA fragments generated by PCR from RNA of NYMV-infected Vero cells, we constructed a rescue plasmid containing the putative full-length NYMV antigenome. As recently described for reverse genetics systems of Borna disease virus and measles virus (9), the full-length NYMV cDNA was inserted into the vector p...

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