Nuclear Transport of the Major Capsid Protein Is Essential for Adeno-Associated Virus Capsid Formation

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.     

Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells.

The three capsid proteins VP1, VP2, and VP3 of the adeno-associated virus type 2 (AAV-2) are encoded by overlapping sequences of the same open reading frame. Separate expression of these proteins by recombinant baculoviruses in insect cells was achieved by mutation of the internal translation initiation codons. Coexpression of VP1 and VP2, VP2 and VP3, and all three capsid proteins and the expression of VP2 alone in Sf9 cells resulted in the production of viruslike particles resembling empty capsids generated during infection of HeLa cells with AAV-2 and adenovirus. These results suggest a requirement for VP2 in the formation of empty capsids. Individual expression of the AAV capsid proteins in HeLa cells showed that VP1 and VP2 accumulate in the cell nucleus and VP3 is distributed between nucleus and cytoplasm. Coexpression of VP3 with the other structural proteins also led to nuclear localization of VP3, indicating that the formation of a complex with VP1 or VP2 is required for accumulation of VP3 in the nucleus.     

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.     

Major and minor capsid proteins of human polyomavirus JC cooperatively accumulate to nuclear domain 10 for assembly into virions

The human polyomavirus JC (JCV) replicates in the nuclei of infected cells. Here we report that JCV virions are efficiently assembled at nuclear domain 10 (ND10), which is also known as promyelocytic leukemia (PML) nuclear bodies. The major capsid protein VP1, the minor capsid proteins VP2 and VP3, and a regulatory protein called agnoprotein were coexpressed from a polycistronic expression vector in COS-7 cells. We found that VP1 accumulated to distinct subnuclear domains in the presence of VP2/VP3 and agnoprotein, while VP1 expressed alone was distributed both in the cytoplasm and in the nucleus. Mutation analysis revealed that discrete intranuclear accumulation of VP1 requires the presence of either VP2 or VP3. However, VP2 or VP3 expressed in the absence of VP1 showed diffuse, not discrete, nuclear localization. The C-terminal sequence of VP2/VP3 contains two basic regions, GPNKKKRRK (cluster 1) and KRRSRSSRS (cluster 2). The deletion of cluster 2 abolished the accumulation of VP1 to distinct subnuclear domains. Deletion of the C-terminal 34 residues of VP2/VP3, including both cluster 1 and cluster 2, caused VP1 to localize both in the cytoplasm and in the nucleus. Using immunoelectron microscopy of cells that coexpressed VP1, VP2/VP3, and agnoprotein, we detected the assembly of virus-like particles in discrete locations along the inner nuclear periphery. Both in oligodendrocytes of the human brain and in transfected cells, discrete nuclear domains for VP1 accumulation were identified as ND10, which contains the PML protein. These results indicate that major and minor capsid proteins cooperatively accumulate in ND10, where they are efficiently assembled into virions.     

Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly

Human polyomavirus JC (JCV) can encode the three capsid proteins VP1, VP2, and VP3, downstream of the agnoprotein in the late region. JCV virions are identified in the nucleus of infected cells. In this study, we have elucidated unique features of JCV capsid formation by using a eukaryotic expression system. Structures of JCV polycistronic late RNAs (M1 to M4 and possibly M5 and M6) generated by alternative splicing were determined. VP1 would be synthesized from M2 RNA, and VP2 and VP3 would be synthesized from M1 RNA. The presence of the open reading frame of the agnoprotein or the leader sequence (nucleotides 275 to 409) can decrease the expression level of VP1. VP1 was efficiently transported to the nucleus in the presence of VP2 and VP3 but distributed both in the cytoplasm and in the nucleus in their absence. Mutation analysis indicated that inefficiency in nuclear transport of VP1 is due to the unique structure in the N-terminal sequence, KRKGERK. Within the nucleus, VP1 was localized discretely and identified as speckles in the presence of VP2 and VP3 but distributed diffusely in their absence. These results suggest that VP1 was efficiently transported to the nucleus and localized in the discrete subnuclear regions, possibly with VP2 and VP3. By electron microscopy, recombinant virus particles were identified in the nucleus, and their intranuclear distribution was consistent with distribution of speckles. This system provides a useful model with which to understand JCV capsid formation and the structures and functions of the JCV capsid proteins.     

Low pH-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the VP1 N-terminal sequence (N-VP1), N-VP2 cleavage, and uncoating of the full-length genome

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.     

Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein

VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.     

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.     

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.     

The First Identified Nucleocytoplasmic Shuttling Herpesviral Capsid Protein: Herpes Simplex Virus Type 1 VP19C

VP19C is a structural protein of herpes simplex virus type 1 viral particle, which is essential for assembly of the capsid. In this study, a nuclear export signal (NES) of VP19C is for the first time identified and mapped to amino acid residues 342 to 351. Furthermore, VP19C is demonstrated to shuttle between the nucleus and the cytoplasm through the NES in a chromosomal region maintenance 1 (CRM1)-dependent manner involving RanGTP hydrolysis. This makes VP19C the first herpesviral capsid protein with nucleocytoplasmic shuttling property and adds it to the list of HSV-1 nucleocytoplasmic shuttling proteins.     

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T = 3 capsid protein leads to larger T = 4 capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.     

人多瘤病毒BK株VP1的C端阳电荷残基对病毒样颗粒形成的影响

VP1是人多瘤病毒BK株的主要结构蛋白,使用重组杆状病毒表达系统在体外表达 VP1 可以形成病毒样颗粒(VLP)。为了探讨VP1的C末端阳电荷残基R 281, R 285, K 288, R 290, R 292, K 293, R 294,和 K297 对VLP形成和其结合DNA的影响,我们分别改变将阳电荷残基变成丙氨酸,然后表达 VP1 蛋白。结果发现用丙氨酸替代K 288,R 290,R 292,K 293,R 294后仍能形成VLP, 但与野毒株相比,在 VLP分泌以及衣壳蛋白与细胞DNA的结合方面有差异。有趣的是,R 281被丙氨酸取代后仅在细胞中形成少量的 VLP,而 R 285 被丙氨酸取代后不能形成VLP。该研究证实阳电荷氨基酸残基 R 281 和 R 285 是形成 VLP所必须的,K 288、R 290、R 292、K 293、R 294和K 297则影响VLP和DNA的结合。     

Simian virus 40 agnoprotein facilitates perinuclear-nuclear localization of VP1, the major capsid protein.

The agnoprotein of simian virus 40 (SV40) is a 61-amino-acid protein encoded in the leader of some late mRNAs. In indirect immunofluorescence studies with antisera against SV40 capsid proteins, we show that mutants which make no agnoprotein display abnormal perinuclear-nuclear localization of VP1, the major capsid protein, but not VP2 or VP3, the minor capsid proteins. In wild-type (WT) SV40-infected CV-1P cells, VP1 was found predominantly in the cytoplasm until 36 h postinfection (p.i.), approximately the time that high levels of agnoprotein became detectable under our infection conditions. Thereafter, VP1 localized rapidly to the perinuclear region and to the nucleus. In contrast, in agnoprotein-minus mutant-infected CV-1P cells, perinuclear-nuclear accumulation of VP1 occurred much less efficiently; a significantly greater fraction of cells with predominantly cytoplasmic fluorescence was observed up to 48 h p.i. At 48 and 60 h p.i., more cells with largely perinuclear and little nuclear staining were seen than in WT-infected controls. In similar analyses with stably transfected cell lines constitutively expressing the agnoprotein, VP1 localized to the nucleus before 30 h p.i., regardless of the infecting virus. Delayed nuclear entry of VP1 in a mutant which makes no agnoprotein was also overcome in a revertant which has a second site point mutation in VP1. This suggests that an alteration of VP1 can partially overcome the defect of the agnogene mutation by enhancement of the rate of its own nuclear localization. Taken together, these results indicate that at least one function of the agnoprotein is to enhance the efficiency of perinuclear-nuclear localization of VP1.     

Disassociation of the SV40 Genome from Capsid Proteins Prior to Nuclear Entry

ABSTRACT: BACKGROUND: Previously, we demonstrated that input SV40 particles undergo a partial disassembly in the endoplasmic reticulum, which exposes internal capsid proteins VP2 and VP3 to immunostaining. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection, as well as to detection by an ethynyl-2-deoxyuridine (EdU)-based chemical reaction. The cytoplasmic partially disassembled SV40 particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. FINDINGS: In the current study, we asked where in the cell the SV40 genome might disassociate from capsid components. We observed partially disassembled input SV40 particles around the nucleus and, beginning at 12 hours post-infection, 5-Bromo-2-deoxyuridine (BrdU)-labeled parental SV40 DNA in the nucleus, as detected using anti-BrdU antibodies. However, among the more than 1500 cells examined, we never detected input VP2/VP3 in the nucleus. Upon translocation of the BrdU-labeled SV40 genomes into nuclei, they were transcribed and, thus, are representative of productive infection CONCLUSIONS: Our findings imply that the SV40 genome disassociates from the capsid proteins before or at the point of entry into the nucleus, and then enters the nucleus devoid of VP2/3..     

Nuclear transport of trimeric assembly intermediates exerts a morphogenetic control on the icosahedral parvovirus capsid

The connection between nuclear transport and morphogenesis of a large macromolecular entity has been investigated using the karyophylic capsid of the parvovirus minute virus of mice (MVM) as a model. The VP1 (82 kDa) and VP2 (63 kDa) proteins forming the T = 1 icosahedral MVM capsid at the respective 1:5 molar ratio of synthesis, could be covalently cross-linked with dimethyl suberimidate into two types of oligomeric assemblies, which were present at stoichiometric amounts in infected cell extracts and purified viral particles. The larger species contained VP1 and corresponded in size (200 kDa) to a heterotrimer of one VP1 and two VP2 subunits. The smaller species contained VP2 only and corresponded in size (180 kDa) to a homotrimer. The introduction of bulky residues or the truncation of side-chains involved in multiple interactions at the interfaces between trimers of VPs in the MVM capsid, produced the accumulation of trimeric intermediates that were competent in nuclear translocation but not in capsid assembly. These results indicate that MVM maturation proceeds by cytoplasmic oligomerization of the capsid subunits into two types of trimers, which are the assembly intermediates competent to translocate across the nuclear membrane. Consistent with this conclusion, mutations at basic residues that inactivate a previously identified beta-stranded nuclear localization motif, which notably are not involved in inter or intra-subunit contacts, led to cytoplasmic retention of the two types of trimers, with no evidence for other assembly intermediates. Although a fraction of the VP1-containing trimers were translocated into the nucleus driven by the conventional nuclear transport signal of VP1 N terminus, their further assembly in the absence of the VP2-only trimers yielded large molecular mass amorphous aggregates. Therefore, the nuclear transport stoichiometry of assembly intermediates may exert a morphogenetic quality control on macromolecular complexes like the MVM capsid.     

Characterization of the DNA binding properties of polyomavirus capsid protein.

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.     

Intracellular localization of adeno-associated viral proteins expressed in insect cells

Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells.     

Cooperation of structural proteins during late events in the life cycle of polyomavirus.

The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1.     

Subcellular compartmentalization of adeno-associated virus type 2 assembly.

Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformation-specific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.     

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins.

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.