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..     

SV40 VP2 and VP3 insertion into ER membranes is controlled by the capsid protein VP1: implications for DNA translocation out of the ER

Nonenveloped viruses such as Simian Virus 40 (SV40) exploit established cellular pathways for internalization and transport to their site of penetration. By analyzing mutant SV40 genomes that do not express VP2 or VP3, we found that these structural proteins perform essential functions that are regulated by VP1. VP2 significantly enhanced SV40 particle association with the host cell, while VP3 functioned downstream. VP2 and VP3 both integrated posttranslationally into the endoplasmic reticulum (ER) membrane. Association with VP1 pentamers prevented their ER membrane integration, indicating that VP1 controls the function of VP2 and VP3 by directing their localization between the particle and the ER membrane. These findings suggest a model in which VP2 aids in cell binding. After capsid disassembly within the ER lumen, VP3, and perhaps VP2, oligomerizes and integrates into the ER membrane, potentially creating a viroporin that aids in viral DNA transport out of the ER.     

Caveolar endocytosis of simian virus 40 is followed by brefeldin A-sensitive transport to the endoplasmic reticulum, where the virus disassembles

Simian virus 40 (SV40) enters cells by atypical endocytosis mediated by caveolae that transports the virus to the endoplasmic reticulum (ER) instead of to the endosomal-lysosomal compartment, which is the usual destination for viruses and other cargo that enter by endocytosis. We show here that SV4O is transported to the ER via an intermediate compartment that contains beta-COP, which is best known as a component of the COPI coatamer complexes that are required for the retrograde retrieval pathway from the Golgi to the ER. Additionally, transport of SV40 to the ER, as well as infection, is sensitive to brefeldin A. This drug acts by specifically inhibiting the ARF1 GTPase, which is known to regulate assembly of COPI coat complexes on Golgi cisternae. Moreover, some beta-COP colocalizes with intracellular caveolin-1, which was previously shown to be present on a new organelle (termed the caveosome) that is an intermediate in the transport of SV40 to the ER (L. Pelkmans, J. Kartenbeck, and A. Helenius, Nat. Cell Biol. 3:473-483, 2001). We also show that the internal SV40 capsid proteins VP2 and VP3 become accessible to immunostaining starting at about 5 h. Most of that immunostaining overlays the ER, with some appearing outside of the ER. In contrast, immunostaining with anti-SV40 antisera remains confined to the ER.     

The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence.

A cDNA fragment coding for poliovirus capsid polypeptide VP1 was inserted into a simian virus 40 (SV40) genome in the place of the SV40 VP1 gene and fused in phase to the 3' end of the VP2-VP3 genes. Simian cells were infected with the resulting hybrid virus in the presence of an early SV40 mutant used as a helper. Indirect immunofluorescence analysis of the infected cells using anti-poliovirus VP1 immune serum revealed that the SV40/poliovirus fusion protein was located inside the cell nucleus. Deletions of various lengths were generated in the SV40 VP2-VP3 portion of the hybrid gene using BAL31 nuclease. The resulting virus genomes expressed spliced fusion proteins whose intracellular location was either intranuclear or intracytoplasmic, depending on the presence or absence of VP2 amino acid residues 317 to 323 (Pro-Asn-Lys-Lys-Lys-Arg-Lys). This was confirmed by site-directed mutagenesis of the Lys residue at position 320. Modification of Lys-320 into either Thr or Asn abolished the nuclear accumulation of the fusion protein. It is concluded that at least part of the sequence of VP2 amino acids 317 to 323 allows VP2 and VP3 to remain stably located inside the cell nucleus. The proteins are most probably transported from the cell cytoplasm to the cell nucleus by interaction, with VP1 acting as a carrier.     

A large and intact viral particle penetrates the endoplasmic reticulum membrane to reach the cytosol

Non-enveloped viruses penetrate host membranes to infect cells. A cell-based assay was used to probe the endoplasmic reticulum (ER)-to-cytosol membrane transport of the non-enveloped SV40. We found that, upon ER arrival, SV40 is released into the lumen and undergoes sequential disulfide bond disruptions to reach the cytosol. However, despite these ER-dependent conformational changes, SV40 crosses the ER membrane as a large and intact particle consisting of the VP1 coat, the internal components VP2, VP3, and the genome. This large particle subsequently disassembles in the cytosol. Mutant virus and inhibitor studies demonstrate VP3 and likely the viral genome, as well as cellular proteasome, control ER-to-cytosol transport. Our results identify the sequence of events, as well as virus and host components, that regulate ER membrane penetration. They also suggest that the ER membrane supports passage of a large particle, potentially through either a sizeable protein-conducting channel or the lipid bilayer.     

The primary sequence of the late region of polyoma virus DNA II. The expression of the late genes and comparison with DNA sequences of SV40 and BKV.

The nucleotide sequence of the late region of the polyoma virus genome has been deduced, which codes for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. The amino acid sequence of VP1 predicted from the nucleotide sequence is in good agreement with the partial N-terminal sequence 1 and amino acid composition of VP1 2,3. When both nucleotide and amono acid sequences are compared with their counterparts in the related viruses, SV40 4,5 and BKV (R. Young, personal communication), extensive homologies are found along the entire regions of the viral genes. Maximum homologies appear to occur in the regions which code for the C-terminal of VP1, on the contrary of the result of heteroduplex analysis 6 with 6 with SV40 and polyoma virus DNAs.     

Calcium Bridge Triggers Capsid Disassembly in the Cell Entry Process of Simian Virus 40

The calcium bridge between the pentamers of polyoma viruses maintains capsid metastability. It has been shown that viral infection is profoundly inhibited by the substitution of lysine for glutamate in one calcium-binding residue of the SV40 capsid protein, VP1. However, it is unclear how the calcium bridge affects SV40 infectivity. In this in vitro study, we analyzed the influence of host cell components on SV40 capsid stability. We used an SV40 mutant capsid (E330K) in which lysine had been substituted for glutamate 330 in protein VP1. The mutant capsid retained the ability to interact with the SV40 cellular receptor GM1, and the internalized mutant capsid accumulated in caveolin-1-mediated endocytic vesicles and was then translocated to the endoplasmic reticulum (ER) region. However, when placed in ER-rich microsome, the mutant capsid retained its spherical structure in contrast to the wild type, which disassembled. Structural analysis of the mutant capsid with cryo-electron microscopy and image reconstruction revealed altered pentamer coordination, possibly as a result of electrostatic interaction, although its overall structure resembled that of the wild type. These results indicate that the calcium ion serves as a trigger at the pentamer interface, which switches on capsid disassembly, and that the failure of the E330K mutant capsid to disassemble is attributable to an inadequate triggering system. Our data also indicate that calcium depletion-induced SV40 capsid disassembly may occur in the ER region and that this is essential for successful SV40 infection.     

Nuclear localization of poliovirus capsid polypeptide VP1 expressed as a fusion protein with SV40-VP1

The poliovirus cDNA fragment coding for capsid polypeptide VP1 was inserted between the EcoRI and BamHI sites of SV40 DNA, generating a chimaeric gene in which the sequence of the 302 amino acids (aa) of poliovirus capsid polypeptide VP1 was placed downstream from that of the 94 N-terminal aa of SV40 capsid polypeptide VP1. The resulting defective, hybrid virus, SV40-delta 1 polio, was propagated in CV1 cells using an early SV40 mutant, am404, as a helper. Cells doubly infected by SV40-delta 1 polio and am404 expressed a 50-kDal fusion protein which was specifically immunoprecipitated by polyclonal and/or monoclonal antibodies raised against poliovirus capsids or against poliovirus polypeptide VP1. Examination of the infected cells by immunofluorescence after staining with anti-poliovirus VP1 immune sera revealed that the fusion protein was mostly located in the intra- and perinuclear space of the cells, in contrast to the exclusively intracytoplasmic location of genuine poliovirus VP1 polypeptide that was observed in poliovirus-infected cells. This suggests that the N-terminal part of the SV40-VP1 polypeptide could contain an important sequence element acting as a migration signal for the transport of proteins from the cytoplasm to the nucleus.     

Two forms of VP7 are involved in assembly of SA11 rotavirus in endoplasmic reticulum

Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.     

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.     

Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup.

An antigenic determinant common to the major capsid polypeptide (VP1) of simian virus 40 (SV40) and polyoma virus is described. Antisera prepared against intact viral particles reacted only with cells infected with the homologous virus by immunofluorescence tests (IF). However, antisera prepared against disrupted SV40 particles reacted in IF with both polyoma- and SV40-infected permissive cells. The cross-reaction with polyoma was localized to VP1 by the following evidence. (i) The IF cross-reaction was inhibited by preincubation of the antiserum with purified SV40 VP1; (ii) purified radiolabeled polyoma VP1 was precipitated by the cross-reactive serum, and this reaction was inhibited by unlabeled SV40 VP1; (iii) other antisera prepared against purified SV40 VP1 or polyoma VP1 reacted in IF with both SV40- and polyma-infected permissive cells. These cross-reacting antisera also reacted in IF with permissive cells infected with BK virus, rabbit kidney vacuolating virus, and the stumptailed macaque virus, suggesting that all members of the polyoma-SV40 subgroup share a common antigenic determinant located in their major capsid polypeptides.     

A simian virus 40-encoded protein of Mr 74,000 daltons is structurally related to the capsid proteins of the virus

We have demonstrated the synthesis of a 74,000-dalton protein (74K protein) in African green monkey kidney cells infected with simian virus (SV)40. The 74K protein was detected late during the lytic cycle. Its synthesis was inhibited by arabinosyl cytosine as was the synthesis of the capsid proteins. Monospecific antibodies raised against VP1 and VP3 precipitated the structural proteins and the 74K protein. The 74K protein was not found in purified virions. Tryptic peptide analysis demonstrated that the 74K protein shares methionine- and serine-containing peptides with VP1 and VP3 and thus is structurally related to the capsid proteins.     

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.     

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.     

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.     

The abundant nuclear enzyme PARP participates in the life cycle of simian virus 40 and is stimulated by minor capsid protein VP3

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.     

Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity

The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.     

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.     

Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3

The capsid of SV40 is regarded as a potential nano-capsule for delivery of biologically active materials. The SV40 capsid is composed of 72 pentamers of the VP1 major capsid protein and 72 copies of the minor coat proteins VP2/3. We have previously demonstrated that, when expressed in insect Sf9 cells by the baculovirus system, VP1 self-assembles into virus-like particles (VP1-VLPs), which are morphologically indistinguishable from the SV40 virion and can be easily purified. Here, we show that heterologous proteins fused to VP2/3 can be efficiently incorporated into the VP1-VLPs. Using EGFP as a model protein, we have optimized this encapsulation system and found that fusion to the C-terminus of VP2/3 is preferable and that the C-terminal VP1-interaction domain of VP2/3 is sufficient for incorporation into VLPs. The VLPs encapsulating EGFP retain the ability to attach to the cell surface and enter the cells. Using this system, we have encapsulated yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that converts 5-fluorocytosine to 5-fluorouracil, into VLPs. When CV-1 cells are challenged by the yCD-encapsulating VLPs, they become sensitive to 5-fluorocytosine-induced cell death. Therefore, proteins of interest can be encapsulated in VP1-VLPs by fusion to VP2/3 and successfully delivered to cells.