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.     

Nuclear localization of avian polyomavirus structural protein VP1 is a prerequisite for the formation of virus-like particles

Virions of polyomaviruses consist of the major structural protein VP1, the minor structural proteins VP2 and VP3, and the viral genome associated with histones. An additional structural protein, VP4, is present in avian polyomavirus (APV) particles. As it had been reported that expression of APV VP1 in insect cells did not result in the formation of virus-like particles (VLP), the prerequisites for particle formation were analyzed. To this end, recombinant influenza viruses were created to (co)express the structural proteins of APV in chicken embryo cells, permissive for APV replication. VP1 expressed individually or coexpressed with VP4 did not result in VLP formation; both proteins (co)localized in the cytoplasm. Transport of VP1, or the VP1-VP4 complex, into the nucleus was facilitated by the coexpression of VP3 and resulted in the formation of VLP. Accordingly, a mutant APV VP1 carrying the N-terminal nuclear localization signal of simian virus 40 VP1 was transported to the nucleus and assembled into VLP. These results support a model of APV capsid assembly in which complexes of the structural proteins VP1, VP3 (or VP2), and VP4, formed within the cytoplasm, are transported to the nucleus using the nuclear localization signal of VP3 (or VP2); there, capsid formation is induced by the nuclear environment.     

Eclipse phase of herpes simplex virus type 1 infection: Efficient dynein-mediated capsid transport without the small capsid protein VP26

Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-DeltaVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-DeltaVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-DeltaVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin.     

Conformational changes in adeno-associated virus type 1 induced by genome packaging

Adeno-associated virus (AAV) is frequently used as a vector for gene therapy. The viral capsid consists of three structural proteins (VP1, VP2, and VP3) that have a common C-terminal core (VP3), with N-terminal extensions of increasing length in VP2 and VP1. The capsid encloses a single-stranded genome of up to 4.7 kb, which is packaged into empty capsids. The N-terminal extension of VP1 carries a phospholipase domain that becomes accessible during infection in the endosomal pathway. We have used cryo-electron microscopy and image reconstruction to determine subnanometer-resolution structures of recombinant AAV1 that has packaged different amounts of a 3. 6-kb recombinant genome. The maps show that the AAV1 capsid undergoes continuous conformational changes upon packaging of the genome. The rearrangements occur at the inner capsid surface and lead to constrictions of the pores at the 5-fold symmetry axes and to subtle movements of the β-sheet regions of the capsid proteins. In fully packaged particles, the genome forms stem-like features that contact the inner capsid surface at the 3-fold symmetry axes. We think that the reorganization of the inner surface has an impact on the viral life cycle during infection, preparing the externalization of phospholipase domains through the pores at the 5-fold symmetry axes and possibly genome release.     

Nuclear export of the nonenveloped parvovirus virion is directed by an unordered protein signal exposed on the capsid surface

It is uncertain whether nonenveloped karyophilic virus particles may actively traffic from the nucleus outward. The unordered amino-terminal domain of the VP2 major structural protein (2Nt) of the icosahedral parvovirus minute virus of mice (MVM) is internal in empty capsids, but it is exposed outside of the shell through the fivefold axis of symmetry in virions with an encapsidated single-stranded DNA genome, as well as in empty capsids subjected to a heat-induced structural transition. In productive infections of transformed and normal fibroblasts, mature MVM virions were found to efficiently exit from the nucleus prior to cell lysis, in contrast to the extended nuclear accumulation of empty capsids. Newly formed mutant viruses lacking the three phosphorylated serine residues of 2Nt were hampered in their exit from the human transformed NB324K nucleus, in correspondence with the capacity of 2Nt to drive microinjected phosphorylated heated capsids out of the nucleus. However, in normal mouse A9 fibroblasts, in which the MVM capsid was phosphorylated at similar sites but with a much lower rate, the nuclear exit of virions and microinjected capsids harboring exposed 2Nt required the infection process and was highly sensitive to inhibition of the exportin CRM1 in the absence of a demonstrable interaction. Thus, the MVM virion exits the nucleus by accessing nonconventional export pathways relying on cell physiology that can be intensified by infection but in which the exposure of 2Nt remains essential for transport. The flexible 2Nt nuclear transport signal may illustrate a common structural solution used by nonenveloped spherical viruses to propagate in undamaged host tissues.     

Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus

Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A(2) catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.     

Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies

The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.     

Complementary roles of multiple nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice during assembly and onset of infection

This report describes the distribution of conventional nuclear localization sequences (NLS) and of a beta-stranded so-called nuclear localization motif (NLM) in the two proteins (VP1, 82 kDa; VP2, 63 kDa) forming the T=1 icosahedral capsid of the parvovirus minute virus of mice (MVM) and their functions in viral biogenesis and the onset of infection. The approximately 10 VP1 molecules assembled in the MVM particle harbor in its 142-amino-acid (aa) N-terminal-specific region four clusters of basic amino acids, here called BC1 (aa 6 to 10), BC2 (aa 87 to 90), BC3 (aa 109 to 115), and BC4 (aa 126 to 130), that fit consensus NLS and an NLM placed toward the opposite end of the polypeptide (aa 670 to 680) found to be necessary for VP2 nuclear uptake. Deletions and site-directed mutations constructed in an infectious MVM plasmid showed that BC1, BC2, and NLM are cooperative nuclear transport sequences in singly expressed VP1 subunits and that they conferred nuclear targeting competence on the VP1/VP2 oligomers arising in normal infection, while BC3 and BC4 did not display nuclear transport activity. Notably, VP1 proteins mutated at BC1 and -2, and particularly with BC1 to -4 sequences deleted, induced nuclear and cytoplasmic foci of colocalizing conjugated ubiquitin that could be rescued from the ubiquitin-proteasome degradation pathway by the coexpression of VP2 and NS2 isoforms. These results suggest a role for VP2 in viral morphogenesis by assisting cytoplasmic folding of VP1/VP2 subviral complexes, which is further supported by the capacity of NLM-bearing transport-competent VP2 subunits to recruit VP1 into the nuclear capsid assembly pathway regardless of the BC composition. Instead, all four BC sequences, which are located in the interior of the capsid, were absolutely required by the incoming infectious MVM particle for the onset of infection, suggesting either an important conformational change or a disassembly of the coat for nuclear entry of a VP1-associated viral genome. Therefore, the evolutionarily conserved BC sequences and NLM domains provide complementary nuclear transport functions to distinct supramolecular complexes of capsid proteins during the autonomous parvovirus life cycle.     

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.     

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.     

Ultrastructural visualization of individual tegument protein dissociation during entry of herpes simplex virus 1 into human and rat dorsal root ganglion neurons

Herpes simplex virus 1 (HSV-1) enters neurons primarily by fusion of the viral envelope with the host cell plasma membrane, leading to the release of the capsid into the cytosol. The capsid travels via microtubule-mediated retrograde transport to the nuclear membrane, where the viral DNA is released for replication in the nucleus. In the present study, the composition and kinetics of incoming HSV-1 capsids during entry and retrograde transport in axons of human fetal and dissociated rat dorsal root ganglia (DRG) neurons were examined by wide-field deconvolution microscopy and transmission immunoelectron microscopy (TIEM). We show that HSV-1 tegument proteins, including VP16, VP22, most pUL37, and some pUL36, dissociated from the incoming virions. The inner tegument proteins, including pUL36 and some pUL37, remained associated with the capsid during virus entry and transit to the nucleus in the neuronal cell body. By TIEM, a progressive loss of tegument proteins, including VP16, VP22, most pUL37, and some pUL36, was observed, with most of the tegument dissociating at the plasma membrane of the axons and the neuronal cell body. Further dissociation occurred within the axons and the cytosol as the capsids moved to the nucleus, resulting in the release of free tegument proteins, especially VP16, VP22, pUL37, and some pUL36, into the cytosol. This study elucidates ultrastructurally the composition of HSV-1 capsids that encounter the microtubules in the core of human axons and the complement of free tegument proteins released into the cytosol during virus entry.     

The minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting.

The terminal hairpin structures of the DNA of minute virus of mice (MVM) are essential for viral replication. Here we show that the hairpin 3' terminus of MVM replicative-form DNA binds specifically to empty MVM capsids. Binding of the same terminal DNA sequence in its linear double-stranded (extended) conformation was not observed. After heat denaturation and quick cooling of 3'-terminal extended-form fragments, not only the virion strand but also the complementary strand was found to bind to the capsid, presumably because each strand re-formed a similar hairpin structure. No binding affinity for the capsid was found to be associated with hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forming a binding site at the branch point of the two 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single base changes within this site did not affect the binding. In band shift experiments, specific binding to the T structure was demonstrated for VPI but not for VP2.     

Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport

Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 degrees C. After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains.     

C terminus of infectious bursal disease virus major capsid protein VP2 is involved in definition of the T number for capsid assembly

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.     

The major capsid protein VP2 of minute virus of mice (MVM) can form particles which bind to the 3'-terminal hairpin of MVM replicative-form DNA and package single-stranded viral progeny DNA.

The capsids of minute virus of mice (MVM) consist of two closely related proteins, VP1 and VP2. We inactivated the VP1 gene in an infectious clone of MVM DNA by frameshift mutation. After transfection of mutated DNA, capsids consisting of VP2 only were made. They can package negative-strand DNA, and they specifically bind MVM 3'-terminal hairpin DNA.     

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.     

Microtubule-mediated Transport of Incoming Herpes Simplex Virus 1 Capsids to the Nucleus

Herpes simplex virus 1 fuses with the plasma membrane of a host cell, and the incoming capsids are efficiently and rapidly transported across the cytosol to the nuclear pore complexes, where the viral DNA genomes are released into the nucleoplasm. Using biochemical assays, immunofluorescence, and immunoelectron microscopy in the presence and absence of microtubule depolymerizing agents, it was shown that the cytosolic capsid transport in Vero cells was mediated by microtubules. Antibody labeling revealed the attachment of dynein, a minus end–directed, microtubule-dependent motor, to the viral capsids. We propose that the incoming capsids bind to microtubules and use dynein to propel them from the cell periphery to the nucleus.     

Improper Tagging of the Non-Essential Small Capsid Protein VP26 Impairs Nuclear Capsid Egress of Herpes Simplex Virus

To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17(+)) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.     

A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini

The complex infection process of parvoviruses is not well understood so far. An important role has been attributed to a phospholipase A2 domain which is located within the unique N terminus of the capsid protein VP1. Based on the structural difference between adeno-associated virus type 2 wild-type capsids and capsids lacking VP1 or VP2, we show via electron cryomicroscopy that the N termini of VP1 and VP2 are involved in forming globules inside the capsids of empty and full particles. Upon limited heat shock, VP1 and possibly VP2 become exposed on the outsides of full but not empty capsids, which is correlated with the disappearance of the globules in the inner surfaces of the capsids. Using molecular modeling, we discuss the constraints on the release of the globularly organized VP1-unique N termini through the channels at the fivefold symmetry axes outside of the capsid.