Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L ؋ T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the 2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the 2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of ␣-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of 2 with the changes Pro-208 to Ser in a background of T1L 2 and Ser-208 to Pro in a background of T3D 2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the 2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.

Cells infected with mammalian orthoreoviruses contain large cytoplasmic phase-dense inclusions believed to be the sites of viral replication and assembly, but the morphogenesis, structure, and specific functions of these "viral factories" are poorly understood. Using immunofluorescence microscopy, we found that reovirus nonstructural protein NS expressed in transfected cells forms inclusions that resemble the globular viral factories formed in cells infected with reovirus strain type 3 Dearing from our laboratory (T3D N ). In the transfected cells, the formation of NS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller NS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of NS and reovirus protein 2 from a different strain, type 1 Lang (T1L), which forms filamentous viral factories, altered the distributions of both proteins. In cotransfected cells, the two proteins colocalized in thick filamentous structures. After nocodazole treatment, many small dispersed globular inclusions containing NS and 2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the 2 protein from T3D N also colocalized with NS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing NS and 2 protein from T3D N and the filamentous structures containing NS and 2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirusinfected cells, which is determined by the 2-encoding M1 genome segment. We found that the first 40 amino acids of NS are required for colocalization with 2 but not for inclusion formation. Similarly, a fusion of NS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the 2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between NS and NSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of NS. The capacity of NS to form inclusions and to colocalize with 2 in transfected cells suggests a key role for NS in forming viral factories in reovirus-infected cells.The replication and assembly of viruses are often concentrated in specific locations within infected cells, such as on the actin cytoskeleton for human parainfluenza virus type 3 (13), on the outer mitochondrial membranes for flock house virus (24), in cytoplasmic inclusions for vaccinia virus (39), and in nuclear inclusions for herpes simplex virus (32). The nonfusogenic mammalian orthoreoviruses (reoviruses) are believed to replicate and assemble in cytoplasmic phase-dense inclusions in infected cells (31). These inclusions contain viral doublestranded RNA (34), viral proteins (9, 31), partially and fully assembled viral particles (...

Canine parvovirus (CPV) is a host range variant of a feline virus that acquired the ability to infect dogs through changes in its capsid protein. Canine and feline viruses both use the feline transferrin receptor (TfR) to infect feline cells, and here we show that CPV infects canine cells through its ability to specifically bind the canine TfR. Receptor binding on host cells at 37°C only partially correlated with the host ranges of the viruses, and an intermediate virus strain (CPV type 2) bound to higher levels on cells than did either the feline panleukopenia virus or a later strain of CPV. During the process of adaptation to dogs the later variant strain of CPV gained the ability to more efficiently use the canine TfR for infection and also showed reduced binding to feline and canine cells compared to CPV type 2. Differences on the top and the side of the threefold spike of the capsid surface controlled specific TfR binding and the efficiency of binding to feline and canine cells, and these differences also determined the cell infection properties of the viruses.Canine parvovirus (CPV) emerged in 1978 as the cause of new enteric and myocardial diseases in dogs. The new virus spread globally in a pandemic of disease during 1978 and has since remained endemic in dogs throughout the world (27, 43). The 1978 strain of CPV (termed CPV type 2) was a new virus infecting dogs since there is no serological or other evidence for infection of dogs by a related virus prior to the mid-1970s (27). Phylogenetic analysis shows that all CPV isolates were descended from a single ancestor which emerged during the mid-1970s, which was closely related to the long-known feline panleukopenia virus (FPV) which infects cats, mink, and raccoons but not dogs or cultured dog cells (43). FPV and CPV isolates differ by as little as 0.5% in DNA sequence, and the characteristic properties of CPV type 2 are controlled by a small number of changes in the capsid surface. Two differences between FPV and CPV changed VP2 residues 93 from Lys to Asn and 323 from Asp to Asn, and those changes alone could introduce the canine host range, a CPV-specific antigenic epitope, and a difference in the pH dependence of hemagglutination into FPV (9, 14). Despite the close relationship to FPV, CPV type 2 isolates did not replicate in cats (42,44), and this host range was determined at least in part by VP2 residues 80, 564, and 568 which are in close proximity in the capsid structure (41). Other mutations in the same structural region of CPV type 2 were selected by passage in cat cells (VP2 residue 300 from Ala to Asp), and these reduced the infection of canine cells, as did closely adjacent changes in in vitro prepared mutants (VP2 residue 299 Gly to Glu) (18,26).Host range-controlling residues are located on a raised region of the capsid that surrounds the threefold axis (the threefold spike) (9, 46). VP2 residues 93 and 323 are found near the top of that structure, whereas residues 299 and 300, and changes controlling feline host range, are all on a ridge ...

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are important pathogens of dogs and cats. CPV is a new virus of dogs that first appeared in 1978, having arisen as a variant of a virus that infected cats or a related carnivore (31). CPV and FPV are over 99% identical in DNA sequence, but they differ in host range (29,30). Both viruses can infect feline and mink cells in tissue culture, but only CPV can efficiently infect cultured canine cells (30). FPV infection of dogs is restricted to certain cells of the bone marrow and thymus (30). The molecular determinants of CPV host range have been mapped to three regions on the surface of the capsid structure. Single amino acid changes in these regions lead to loss of the ability of CPV to infect canine, but not feline, cells (8,19). Mutation of residues Asn933Asp and Asn3233Asp in the VP2 capsid protein of FPV to the corresponding amino acids found in the VP2 protein of CPV allows that mutant to infect dog cells (8). The surface location of these host range determinants suggests that host range may be determined by the ability to bind a cell surface receptor or other cellular ligand (1).During natural infections, CPV and FPV infect actively dividing cells of the lymphopoietic system and the crypt cells of the intestine (reviewed in reference 22). Initial virus replication occurs in the oropharyngeal lymphoid tissue, and the virus then spreads hematogenously to other lymphoid organs and the intestine. Autonomous parvoviruses (including CPV and FPV) can replicate only in mitotically active cells during the S phase of the cell cycle (9), and so the target organs in vivo are those that contain actively dividing cell populations.

Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein 1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments 1N (4 kDa; myristoylated) and 1C (72 kDa). In this study, we found that 1 cleavage to yield 1N and 1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective 1 (asparagine 3 alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including 1 conformational change and 1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin ␣-sarcin). In addition, after these particles were allowed to enter cells, the ␦ region of 1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genomedeficient particle, the recoated particles with cleavage-defective 1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of 1N/1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal 1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein NS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with NS. We found that all three of these proteins-1, 2, and 2-localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with NS, on the other hand, each core surface protein colocalized with NS in globular inclusions, supporting the initial hypothesis. We also found that 1, 2, and 2 each localized to filamentous inclusions formed upon the coexpression of NS and 2, a structurally minor core protein that associates with microtubules. The first 40 residues of NS, which are required for association with 2 and the RNA-binding nonstructural protein NS, were not required for association with any of the three core surface proteins. When coexpressed with 2 in the absence of NS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with 2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of NS that had been preformed in those cells, providing evidence that NS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins NS and 2 in this process.

Cell entry by reoviruses requires a large, transcriptionally active subvirion particle to gain access to the cytoplasm. The features of this particle have been the subject of debate, but three primary candidates-the infectious subvirion particle (ISVP), ISVP*, and core particle forms-that differ in whether putative membrane penetration protein 1 and adhesin 1 remain particle bound have been identified. Experiments with antibody reagents in this study yielded new information about the steps in particle disassembly during cell entry. Monoclonal antibodies specific for the ␦ region of 1 provided evidence for a conformational change in 1 and for release of the ␦ proteolytic fragment from entering particles. Antiserum raised against cores provided evidence for entry-related changes in particle structure and identified entering particles that largely lack the ␦ fragment inside cells. Antibodies specific for 1 showed that it is also largely shed from entering particles. Limited coimmunostaining with markers for late endosomes and lysosomes indicated the particles lacking ␦ and 1 did not localize to those subcellular compartments, and other observations suggested that both the particles and free ␦ were released into the cytoplasm. Essentially equivalent findings were obtained with native ISVPs and highly infectious recoated particles containing wild-type proteins. Poorly infectious recoated particles containing a hyperstable mutant form of 1, however, showed no evidence for the in vitro and intracellular changes in particle structure normally detected by antibodies, and these particles instead accumulated in late endosomes or lysosomes. Recoated particles with hyperstable 1 were also ineffective at mediating erythrocyte lysis in vitro and promoting ␣-sarcin coentry and intoxication of cells in cultures. Based on these and other findings, we propose that ISVP* is a transient intermediate in cell entry which mediates membrane penetration and is then further uncoated in the cytoplasm to yield particles, resembling cores, that largely lack the ␦ fragment of 1.The entry of animal viruses into host cells is accompanied by proteolytic cleavage, protein conformational changes, and/or protein shedding events that result in partial or complete disassembly of entering particles. One key aspect of disassembly is the conversion of virions, which often have greater stability in the aqueous environments between cells, to particle forms with more hydrophobic surfaces that can interact with a cellular lipid bilayer and mediate the passage of viral components into the cytoplasm. Other key elements include the activation of viral particles or nucleocapsids for genome transcription, translation, and/or targeting to particular subcellular sites. We have been studying the disassembly cascade and cell entry mechanisms used by a group of large nonenveloped viruses, the mammalian orthoreoviruses (reoviruses).Reoviruses belong to the family Reoviridae, an evolutionarily divergent group of double-stranded RNA viruses whose human-infecting members...

Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a ϳ40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to ␣-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components.