Proteins of viral and cellular origin bind to the Aleutian disease virus (ADV) DNA 3'-terminal hairpin: presentation of a scheme for encapsidation of ADV DNA.

We have observed the binding of viral and cellular proteins to the Aleutian disease virus (ADV) 3' terminus of replicative-form DNA. Gel retardation assays showed specific band shifts produced by whole-cell extracts from either ADV-infected or uninfected cells, as well as band reduction produced by ADV capsids. In all cases, binding was confined to the turnaround, T-shaped terminal form; no binding to the extended conformation of replicative-form DNA was detected. This indicates the importance of the T-shaped secondary structure in protein recognition. We have previously reported the binding of a 3'-terminal ADV DNA restriction fragment to the ADV capsid protein VP1 (K. Willwand and O.-R. Kaaden, Virology 166:52-57, 1988). Here we show that the region between nucleotides 14 and 102 on the ADV genome is required for binding. It is suggested that the VP1-DNA interaction mediates the binding of ADV DNA to empty viral capsids and that this is followed by displacement synthesis and packaging of ADV progeny DNA. A scheme for the possible mechanism of this process is presented.     

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.     

Ultrastructural studies of H-1 parvovirus replication. V. Immunocytochemical demonstration of separate chromatin-associated and inclusion-associated antigens.

Electron microscopy and immunocytochrome c staining were used to define the phenotypes of several temperature-sensitive (ts) H-1 mutants. They were classified into three separate groups based on the properties of their capsids at the restrictive temperature (rT): (class 1) ts2 did not assemble capsids but produced spherical and irregular amorphous inclusions; (class 2) ts1 and ts7 exclusively synthesized empty particles which all aggregated and crystallized; and (class 3) ts8 and ts10 formed noncrystalline aggregates of empty virions, but many individual full, as well as empty, capsids were associated with euchromatin. Synthesis of progeny DNA and hemagglutinin at rT were normal for class 3 mutants, but defective for those in classes 1 and 2. The immunospecific staining patterns of these mutants indicated that the H-1 capsid proteins probably form two separate intranuclear antigens: (i) a thermostable chromatin-associated antigen present in proteins that have not formed capsids and are concentrated on heterochromatin and nucleolar-associated chromatin and (ii) a thermolabile inclusion-associated antigen found in the proteins of assembled empty capsids that compose H-1 inclusions.     

Assemblons: nuclear structures defined by aggregation of immature capsids and some tegument proteins of herpes simplex virus 1.

In cells infected with herpes simplex virus 1 (HSV-1), the viral proteins ICP5 (infected-cell protein 5) and VP19c (the product of UL38) are associated with mature capsids, whereas the same proteins, along with ICP35, are components of immature capsids. Here we report that ICP35, ICP5, and UL38 (VP19c) coalesce at late times postinfection and form antigenically dense structures located at the periphery of nuclei, close to but not abutting nuclear membranes. These structures were formed in cells infected with a virus carrying a temperature-sensitive mutation in the UL15 gene at nonpermissive temperatures. Since at these temperatures viral DNA is made but not packaged, these structures must contain the proteins for immature-capsid assembly and were therefore designated assemblons. These assemblons are located at the periphery of a diffuse structure composed of proteins involved in DNA synthesis. This structure overlaps only minimally with the assemblons. In contrast, tegument proteins were located in asymmetrically distributed structures also partially overlapping with assemblons but frequently located nearer to nuclear membranes. Of particular interest is the finding that the UL15 protein colocalized with the proteins associated with viral DNA synthesis rather than with assemblons, suggesting that the association with DNA may take place during its synthesis and precedes the involvement of this protein in packaging of the viral DNA into capsids. The formation of three different compartments consisting of proteins involved in viral DNA synthesis, the capsid proteins, and tegument proteins suggests that there exists a viral machinery which enables aggregation and coalescence of specific viral protein groups on the basis of their function.     

Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine.

Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.     

Polypeptide Composition of Poliovirions, Naturally Occurring Empty Capsids, and 14S Precursor Particles

The three serotypes of poliovirus were compared with respect to their polypeptide composition. Type 1, 2, and 3 strains were clearly different from each other in the electrophoretic mobilities of their larger structural polypeptides. Some of the viral polypeptides formerly identified as single peaks (e.g., VP 2) were shown to contain multiple components, indicating that purified virions contain at least six polypeptides. Three type 1 strains were indistinguishable in their viral polypeptides. A quantitative estimate was made of the polypeptide composition of the type 1 Mahoney poliovirion, as well as of naturally occurring empty capsids and 14S precursor particles. The data are discussed in light of the antigenic differences among polioviruses and the possible modes of virion morphogenesis.     

Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids.

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.     

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.     

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.     

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.     

Temporal association of the herpes simplex virus genome with histone proteins during a lytic infection

Previous work has determined that there are nucleosomes on the herpes simplex virus (HSV) genome during a lytic infection but that they are not arranged in an equally spaced array like in cellular DNA. However, like in cellular DNA, the promoter regions of several viral genes have been shown to be associated with nucleosomes containing modified histone proteins that are generally found associated with actively transcribed genes. Furthermore, it has been found that the association of modified histones with the HSV genome can be detected at the earliest times postinfection (1 h postinfection) and increases up to 3 h postinfection. However from 3 h to 6 h postinfection (the late phase of the replication cycle), the association decreases. In this study we have examined histone association with promoter regions of all kinetic classes of genes. This was done over the time course of an infection in Sy5y cells using sucrose gradient sedimentation, bromodeoxyuridine labeling, chromatin immunoprecipitation assays, Western blot analysis, trypsin and DNase digestion, and quantitative real-time PCR. Because no histones were detected inside HSV type 1 capsids, the viral genome probably starts to associate with histones after being transported from infecting virions into the host nucleus. Promoter regions of all gene classes (immediate early, early, and late) bind with histone proteins at the start of viral gene expression. However, after viral DNA replication initiates, histones appear not to associate with newly synthesized viral genomes.     

Characterization of the mRNA''s for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.     

Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells.

Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP proteins formed hollow intranuclear shells around the inclusions. Later, nuclei had irregular outlines and were virtually free of ADV products. In these cells, inclusions of viral DNA with or without associated NS protein were embedded in cytoplasmic VP protein. These findings implied that ADV replication within an infected cell is regulated spatially as well as temporally.     

ATP-Dependent localization of the herpes simplex virus capsid protein VP26 to sites of procapsid maturation

The herpes simplex virus type 1 (HSV-1) capsid shell is composed of four major polypeptides, VP5, VP19c, VP23, and VP26. VP26, a 12-kDa polypeptide, is associated with the tips of the capsid hexons formed by VP5. Mature capsids form upon angularization of the shell of short-lived, fragile spherical precursors termed procapsids. The cold sensitivity and short-lived nature of the procapsid have made its isolation and biochemical analysis difficult, and it remains unclear whether procapsids contain bound VP26 or whether VP26 is recruited following shell angularization. By indirect immunocytochemical analysis of virally expressed VP26 and by direct visualization of a transiently expressed VP26-green fluorescent protein fusion, we show that VP26 fails to specifically localize to intranuclear procapsids accumulated following incubation of the temperature-sensitive HSV mutant tsProt.A under nonpermissive conditions. However, following a downshift to the permissive temperature, which allows procapsid maturation to proceed, VP26 was seen to concentrate at intranuclear sites which also contained epitopes specific to mature, angularized capsids. Like the formation of these epitopes, the association of VP26 with maturing capsids was blocked in a reversible fashion by the depletion of intracellular ATP. We conclude that unlike the other major capsid shell proteins, VP26 is recruited in an ATP-dependent fashion after procapsid maturation begins.     

Structural polypeptides of rabbit, bovine, and human papillomaviruses.

The number and apparent molecular weight of the structural polypeptides of Shope rabbit papilloma virus (RPV), bovine papilloma virus (BPV), and human papilloma virus (HPV) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Up to 10 polypeptides were detected in highly purified BPV and HPV full particles; a close homology was found between the polypeptide composition of both viruses. Purified RPV virions gave a similar polypeptide pattern. The main components of the three papillomaviruses are the major polypeptide (VP1) with a mol wt of approximately 54,000 and the three smaller polypeptides (VP8, 9, 10) with mol wt of about 16,500, 15,500 and 12,500, respectively. VP8, VP9, and VP10 are never detected in empty capsids. When BPV virions were disrupted with alkaline buffer, the six lower-molecular-weight polypeptides (VP5 to 10) remained associated with viral DNA. This suggests that they are internal components of the virions and that the four higher-molecular-weight polypeptides (VP1 to 4) may represent external components. The polypeptide compositions of BPV and polyoma virus, another papovavirus, have been compared. The number of BPV and polyoma virus components (10 and 6, respectively) and the molecular weight of their major polypeptide (54,000 and 44,500, respectively) are different; however, the three main DNA-associated polypeptides of BPV (VP8, 9, 10) and the three histone-like components of polyoma virus (VP4, 5, 6) were shown to have identical apparent molecular weights. The possibility that some of the minor components of papillomaviruses may be proteolytic degradation products or cell protein contaiminants is discussed.     

In vitro system that synthesizes circular viral DNA of bacteriophage phi X 174.

An extract prepared from Escherichia coli cells infected with phi chi 174 bacteriophage was capable of incorporating dTTP into phage-specific DNAs in vitro. The synthesized DNAs were associated with proteins and sedimented with S values of 20, 50, and 90 in a sucrose gradient sedimentation. DNA isolated from 20S material was open circular replicative form (RF), DNA in 50S material was replicative-form DNA with an extended single-stranded viral DNA that ranged up to one genome in length, and DNA in 90S material consisted of circular and linear single-stranded viral DNA of full genome length and single-stranded viral DNA shorter than full genome length. Pulse and pulse-chase experiments indicated that 90S material derived from 50S material.     

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.     

Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1.

Polyomavirus normally assembles in the nucleus of infected mouse cells. Sf9 insect cells expressing the polyomavirus major capsid protein VP1 were examined by electron microscopy. Capsidlike particles of apparently uniform size were found in the nucleus. Immunogold electron microscopy demonstrated abundant VP1 in the cytoplasm which was not assembled into any recognizable higher-order structure. Cytoplasmic VP1 assembled after the cells were treated with the calcium ionophore ionomycin. Purified VP1 aggregates were shown by negative staining and cryoelectron microscopy to consist predominantly of particles similar to the empty T = 7 viral capsid. Thus, polyomavirus VP1 can assemble in vivo into capsids independent of other viral proteins or DNA. Nuclear assembly may result from increased available calcium in this subcellular compartment.     

Parvovirus replication in normal and transformed human cells correlates with the nuclear translocation of the early protein NS1.

The parvovirus H-1 infection of the normal human diploid fibroblast strain MRC-5 produces a cytopathic effect, but no increase in infectious virus has been observed. Previously, we reported that large amounts of empty capsids are assembled in the nucleus of H-1 infected MRC-5 cells (S. Singer and S. Rhode, in D. Ward and P. Tattersall, ed., Replication of Mammalian Parvoviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1978). The level of viral replicative-form DNA synthesis as shown by metabolic labeling is markedly reduced in these cells. Synthesis of the early protein NS1 is normal or slightly decreased, and the usual amount of the 92,000-molecular-weight (92K) posttranslationally modified NS1 was seen. The second deficient parameter that we have observed in the abortive infection is the nuclear translocation of NS1. In contrast, the simian virus 40-transformed MRC-5 cell line MRC-5 V1 and the simian virus 40-transformed human kidney cell line NB undergo a productive infection by H-1 accompanied by more efficient translocation of NS1 to the nucleus. The results indicate that there is an association between defective translocation of the NS1 rep protein to the nucleus and defective amplification of parvovirus replicative-form DNA. The nuclear translocation of specific proteins seems to be a function that is altered by development or neoplastic transformation.     

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.