Defective Virions of Reovirus

When purified preparations of stock reovirus, type 3, were digested with chymotrypsin, the virions were converted into two different types of particle. These new particles could be separated from each other by isopycnic centrifugation in cesium chloride gradients. One particle banded at a buoyant density of 1.43 g/cm(3), the other at a density of 1.415 g/cm(3). The former particle is termed the heavy (H) particle, the latter is the light (L) particle. The ratio of H/L particles varied between 0.5 and 0.25 in various purified preparations of virus. In electron micrographs, both H and L particles had the appearance and dimensions of viral cores. H particles were infectious for L cells. When plaques formed by stock virus, or by H particles, were picked and propagated in L cells, the majority of the clones gave rise only to H particles on chymotrypsin digestion. On continued serial passage of the clones, virions containing L particles again appeared in the progeny. The simplest explanation of these results was that stock virus was comprised of two populations of virions. One type of virion which contained H particles was infectious, whereas the other, which contained L particles, was not itself infectious and could replicate only in cells coinfected with an H particle virion. Added weight was given to this hypothesis by two observations. First, a small but definite separation of H and L virions could be achieved by isopycnic centrifugation in a gradient of cesium chloride. Second, L particles and virions containing L particles were both shown to lack the largest of the ten segments of double-stranded ribonucleic acid genome. Thus, L particle virions have defective genomes.     

Properties and origins of infectious rhinovirus type 14 particles of different buoyant densities.

Isopycnic centrifugation of rhinovirus type 14 (RV14), purified from infected HeLa or KB cell cultures, into CsCl gradients resolved two bands of infectious virus particles with buoyant density values of 1.409 +/- 0.007 (H virus) and 1.386 +/- 0.004 (L virus) g/ml. Only H virus was detected by incorporation of radiolabeled uridine into viral RNA, and H virus accounted for the majority of infectivity in gradients. H and L virus could not be differentiated by plaque morphology, extent of neutralization by RV14-specific antiserum, or particle size. Electron microscope studies showed that most L-virus particles were associated with an amorphous material. Treatment of L virus with proteolytic enzymes or rebanding L virus in CsCl gradients resulted in recovery of the majority of infectivity as H virus. Virus purified from cell-free fluids from infected HeLa or KB cell cultures banded only as H virus. HeLa cell cultures challenged with purified H virus and harvested at 3 h postinoculation for virus purification yielded only infectious H virus. Both H and L viruses were detected in cell cultures that had been challenged with purified H virus and harvested at 12 h postinoculation. The data suggest that H virus represents progeny virus, whereas L virus represents sequestered infectious virus particles which become associated with an amorphous material and do not enter into viral replicative processes.     

Mechanism of Antiviral Action of Acetone on Rabbitpox Virus Replication

Acetone added to the maintenance medium in a 1% concentration reduces the yield of infectious rabbitpox virus in L-cell monolayer cultures by 90 to 97%. This concentration of the inhibitor is not toxic for cells. It was established that there is an inhibitor-sensitive stage late in the infectious cycle. Acetone exerted no significant influence on production of early viral messenger ribonucleic acid, formation of polyribosomes, deoxyribonucleic acid (DNA) replication, or protein synthesis. In the presence of acetone, the assembly of so-called "acetone particles" occurred. These particles are similar to normal virions by morphological and sedimentation properties but are slightly different from them in buoyant density. The amount of virus-specific DNA and the optical density of the "acetone particles" are the same as those of normal virions despite a 10- to 25-fold difference in the infectivity of the preparations.     

Herpes Simplex Virus Products in Productive and Abortive Infection III. Differentiation of Infectious Virus Derived from Nucleus and Cytoplasm with Respect to Stability and Size

Approximately 67% of infectivity is associated with the nucleus 8 hr after productive infection of HEp-2 cells with herpes simplex virus. Comparison of nuclear and cytoplasmic infectious virus and macromolecular aggregates labeled with (3)H-thymidine or with (3)H-choline revealed the following. (i) Cytoplasmic infectious virus and macromolecular aggregates banded in CsCl at a density corresponding to enveloped nucleocapsids. The virus was relatively stable; there was only 50% loss of infectivity and only 16% of the virions became disaggregated. (ii) Nuclear macromolecular aggregates banded in CsCl solution at a density corresponding to unenveloped nucleocapsids and, moreover, both the infectious virus and aggregates were highly unstable. (iii) In sucrose density gradients, the nuclear macromolecular aggregates and infectivity sedimented as a single band and migrated more slowly than the corresponding cytoplasmic material. (iv) The infectivity of nuclear and cytoplasmic virus is readily inactivated by digestion with phospholipase C and with pronase. We conclude the following. (i) Cytoplasmic virus consists of enveloped nucleocapsids. (ii) Nuclear virus consists of nucleocapsids covered with lipid or partially enveloped. (iii) The molecular integrity of viral lipids is essential for infectivity. (iv) The envelope protects the nucleocapsid and accelerates adsorption to cells; it is not, however, inherently essential for infectivity.     

Mosquito cells infected with vesicular stomatitis virus yield unsialylated virions of low infectivity.

Vesicular stomatitis virus propagated in and released from Aedes albopictus cells had the normal complement of viral proteins; the glycoprotein contained carbohydrate but no sialic acid. These virions had markedly reduced hemagglutinating activity and exhibited a very high ratio of physical particles to infectious virus. In vitro sialylation of vesicular stomatitis virions grown in mosquito cells resulted in a 100-fold increase in both infectivity and hemagglutination titers to levels approaching those of virus grown in BHK-21 cells. These experiments provide an example of host-controlled modification of viral infectivity.     

Complementation of defective reovirus by ts mutants.

Defective reovirions lacking the largest (L-1) of the normal 10 genomic segments grow only in association with helper reovirus. Because of the similarity in properties of defective and infectious virions, separation of the two populations by physical methods has been unseccessful. Controlled digestion of purified virus removes the outer capsomeres of the virions. The resulting core particles containing the viral genome have a buoyant density of 1.43/ml if derived from infectious virions and of 1.415g/ml if they originate in defectives, and this difference permits ready separation of the two types of cores. With the purpose of obtaining a pure population of defective virions, L cells were co-infected with defective cores and a class E temperature-sensitive mutant which has a mutation in an early function. After three serial passages at the permissive temperature (31 C) to build up the defective population, a fourth passage was made at 39 C, the nonpermissive temperature. The virus purified from this passage was predominantly defective; it contained practically no E mutant and had a low background of wild-type virus. Complementation was thus asymmetric; the L-1 function required for growth of defective virus was supplied by the E mutant and is thus a trans-function, while defective virus did not complement the E mutation which is thus in a cis-acting function. Defective virions were indistinguishable from infectious virions except for the absence of the L-1 genomic segment in the defectives. Such defective virions could be complemented at 39 C by class A and B temperature-sensitive mutants, both of which have lesions in late functions.     

Herpes Simplex Virus Products in Productive and Abortive Infection I. Stabilization with Formaldehyde and Preliminary Analyses by Isopycnic Centrifugation in CsCl

Lysates of HEp-2 cells productively infected with herpes simplex virus yielded two bands on isopycnic centrifugation in CsCl gradients, ranging from 1.2 to 1.6 g/cm(3). One band, designated alpha, had a mean buoyant density of 1.27 g/cm(3) and contained herpes virions. Band beta had a mean density of 1.305 g/cm(3) and contained primarily complement-fixing viral antigens and little or no viral deoxyribonucleic acid (DNA). The products banding in the alpha and beta bands were unstable; fivefold or higher amounts were recovered by treating the cell extract with formaldehyde prior to centrifugation. Formaldehyde treatment increased the buoyant density of viral products in both the alpha and beta bands by about 0.015 g/cm(3). In addition, it stabilized hitherto inapparent products, forming a broad band gamma with a density range of 1.37 to 1.45 g/cm(3). The material in the gamma band was heterogeneous; it contained viral DNA, cellular DNA, and viral antigen. Formalinized lysates of DK cells abortively infected with herpes simplex virus yielded a beta band undifferentiated from that formed by extracts of productively infected cells. The gamma band was less dense and narrower. The alpha band was entirely missing.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

Heterogeneity of vesicular stomatitis virus particles: implications for virion assembly

Vesicular stomatitis virus (VSV) particles formed at early times after infection contain only one-third the amount of viral glycoportein (G protein), relative to the major internal structural proteins M and N, as is found in particles released later. These "early" particles also have a lower density in equilibrium sucrose gradients than do those formed later; however, the sedimentation velocity and specific infectivity of these two classes of particles are the same. VSV-infected cells also release virus-like particles which sediment considerably faster than authentic virions and contain a higher-than-normal proportion of the VSV G protein relative to internal VSV proteins. These particles have a reduced specific infectivity but a normal density in sucrose gradients. All classes of VSV virions contain a constant proportion of M and N polypeptides. The ratio of G protein to M or N protein, in contrast, can vary over a sixfold range; this implies that an interaction between a precise number of surface G proteins with either of the underlying M and N proteins is not a prerequisite for budding of infectious viral particles from the cell surface.     

Virions released from cells transfected with a molecular clone of human T-cell leukemia virus type I give rise to primary and secondary infections of T cells.

The ability of molecular clones of human T-cell leukemia virus type I (HTLV-I) to direct the synthesis of infectious virions has not previously been demonstrated. An HTLV-I provirus originating from an adult T-cell leukemia patient was cloned into a plasmid vector and is designated pCS-HTLV. This molecular clone was shown to direct the synthesis of viral mRNA and proteins in transiently transfected cells; in addition, virus structural proteins were released into the culture medium. Viral proteins were assembled into virions that sedimented at a buoyant density characteristic of retrovirus particles and whose morphology was verified by electron microscopy. Virions concentrated from transiently transfected cell supernatants were incubated with primary cord blood lymphocytes or with transformed T-cell lines to establish that these particles were infectious. Expression of spliced, viral mRNAs in the T-cell cultures after both primary and secondary infections with cell-free virus revealed that pCS-HTLV encodes an infectious provirus.     

Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl.

Although hepatitis A virus (HAV) released by infected BS-C-1 cells banded predominantly at 1.325 g/cm3 (major component) in CsCl, smaller proportions of infectious virions banded at 1.42 g/cm3 (dense HAV particles) and at 1.27 g/cm3 (previously unrecognized light HAV particles). cDNA-RNA hybridization confirmed the banding of viral RNA at each density, and immune electron microscopy demonstrated apparently complete viral particles in each peak fraction. The ratio of the infectivity (radioimmunofocus assay) titer to the antigen (radioimmunoassay) titer of the major component was approximately 15-fold greater than that of dense HAV particles and 4-fold that of light HAV particles. After extraction with chloroform, the buoyant density of light and major component HAV particles remained unchanged, indicating that the lower density of the light particles was not due to association with lipids. Light particles also banded at a lower density (1.21 g/cm3) in metrizamide than did the major component (1.31 g/cm3). Dense HAV particles, detected by subsequent centrifugation in CsCl, were indistinguishable from the major component when first banded in metrizamide (1.31 g/cm3). However, dense HAV particles recovered from CsCl subsequently banded at 1.37 g/cm3 in metrizamide. Electrophoresis of virion RNA under denaturing conditions demonstrated that dense, major-component, and light HAV particles all contained RNA of similar length. Thus, infectious HAV particles released by BS-C-1 cells in vitro consist of three distinct types which band at substantially different densities in CsC1, suggesting different capsid structures with varied permeability to cesium or different degrees of hydration.     

Human immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity.

Disruption of the vif gene of human immunodeficiency virus (HIV) type 1 affects virus infectivity to various degrees, depending on the T-cell line used. We have concentrated our studies on true phenotypic Vif- mutant particles produced from CEMx174 or H9 cells. In a single round of infection, Vif- virus is approximately 25 (from CEMx174 cells) to 100 (from H9 cells) times less infectious than wild-type virus produced from these cells or than the Vif- mutant produced from HeLa cells. Vif- virions recovered from restrictive cells, but not from permissive cells, are abnormal both in terms of morphology and viral protein content. Notably, they contain much reduced quantities of envelope proteins and altered quantities of Gag and Pol proteins. Although wild-type and Vif- virions from restrictive cells contain similar quantities of viral RNA, no viral DNA synthesis was detectable after acute infection of target cells with phenotypically Vif- virions. To examine the possible role of Vif in viral entry, attempts were made to rescue the Vif- defect in H9 cells by pseudotyping Vif+ and Vif- HIV particles with amphotropic murine leukemia virus envelope. Vif- particles produced in the presence of HIV envelope could not be propagated when pseudotyped. In contrast, when only the murine leukemia virus envelope was present, significant propagation of Vif- HIV particles could be detected. These results demonstrate that Vif is required for proper assembly of the viral particle and for efficient HIV Env-mediated infection of target cells.     

Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity.

The purpose of this study was to identify the herpes simplex virus glycoprotein(s) that mediates the adsorption of virions to cells. Because heparan sulfate moieties of cell surface proteoglycans serve as the receptors for herpes simplex virus adsorption, we tested whether any of the viral glycoproteins could bind to heparin-Sepharose in affinity chromatography experiments. Two glycoproteins, gB and gC, bound to heparin-Sepharose and could be eluted with soluble heparin. In order to determine whether virions devoid of gC or gB were impaired for adsorption, we quantitated the binding of wild-type and mutant virions to cells. We found that at equivalent input concentrations of purified virions, significantly fewer gC-negative virions bound to cells than did wild-type or gB-negative virions. In addition, the gC-negative virions that bound to cells showed a significant delay in penetration compared with wild-type virus. The impairments in adsorption and penetration of the gC-negative virions can account for their reduced PFU/particle ratios, which were found to be about 5 to 10% that of wild-type virions, depending on the host cell. Although gC is dispensable for replication of herpes simplex virus in cell culture, it clearly facilitates virion adsorption and enhances infectivity by about a factor of 10.     

Deoxyribonucleotide Pools and Deoxyribonucleic Acid Synthesis in Mouse Embryo Cells Infected with Three Classes of Polyoma Virus Particles

Polyoma virus particles were purified by equilibrium centrifugation in CsCl. Particles from three regions of the density gradient were examined for infectivity, for their ability to induce expanded pools of deoxyribonucleic acid (DNA) precursors, and for their ability to stimulate the synthesis of DNA. The most infectious population of particles, the virions, having a buoyant density of 1.33 g/ml, gave the greatest stimulation of the DNA-synthesizing apparatus of mouse embryo cells. Empty particles at density 1.29 g/ml had no DNA stimulatory activity. A population of particles of intermediate density, referred to as pseudovirions, was also much less active than virions in stimulating DNA synthesis, and the limited stimulatory activity of the latter fraction may be accounted for by its measured contamination with infective particles.     

Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.     

Restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components.

Noninfectious spikeless particles have been obtained from vesicular stomatitis virus (VSV, Indiana serotype) by bromelain or Pronase treatment. They lack the viral glycoprotein (G) but contain all the other viral components (RNA, lipid, and other structural proteins). Triton-solubilized VSV-Indiana glycoprotein preparations, containing the viral G protein as well as lipids (including phospholipids), have been extracted from whole virus preparations, freed from the majority of the detergent, and used to restore infectivity to spikeless VSV. The infectivity of such particles has been found to be enhanced by poly-L-ornithine but inhibited by Trition or homologous antiserum pretreatment. Heat-denatured glycoprotein preparations were not effective in restoring the infectivity to spikeless VSV. Heterologous glycoprotein preparations from the serologically distinct VSV-New Jersey serotype were equally capable of making infectious entities with VSV-Indiana spikeless particles, and the infectivity of these structures was inhibited by VSV-New Jersey antiserum but not by VSV-Indiana antiserum. Purified, detergent-free glycoprotein selectively solubilized from VSV-Indiana by the dialyzable detergent, octylglucoside, also restored infectivity of spikeless virions of VSV-Indiana and VSV-New Jersey.     

Is progressive multifocal leukoencephalopathy a chronic disease because of defective interfering particles or temperature-sensitive mutants of JC virus?

JC virus was examined for temperature sensitivity and for evidence of defective interfering particles as a means of explaining the slow chronic nature of progressive multifocal leukoencephalopathy (PML). JC virus direct from the brain tissue of seven persons with PML was not temperature sensitive as indicated by in vitro assay at 37 and 39 degrees C. In fact, more cells contained viral antigen at 39 than at 37 degrees C. The amount of infectious virus also was increased at 39 degrees C. Virions isolated directly from diseased brain tissue had a higher buoyant density than did virus from the same PML patient passaged in culture and containing genomic deletions. In contrast to DNA from culture-passed virus, DNA extracted from virions direct from brain tissue was homogeneous in length. In 13 separate cases examined, the viral DNA direct from the brain was homogeneous although variations in length were noted among DNAs from different cases. Restriction enzyme cleavage patterns identified all as JC virus DNA. It was concluded that neither temperature sensitivity nor DI particles can be used to explain the slow, progressive nature of PML.     

Reconstituted G protein-lipid vesicles from vesicular stomatitis virus and their inhibition of VSV infection

The single glycoprotein (G) of vesiclar stomatitis virus (VSV) was isolated in nearly quantitative yield by extraction of the purified virions with 0.05 M octyl-β-D- glucoside (OG) in 0.01 M sodium phosphate, pH 8.0. The extract contained essentially all of the viral phospholipids and glycolipids, and was free of other essentially all of the viral phospholipids and glycolipids, and was free of other viral proteins. Dialysis to remove OG resulted in the formation of G protein-viral lipid vesicles having a lipid-G protein ratio similar to that of the intact virions. The vesicles were 250-1,000 A in diameter, with a “fuzzy” external layer also similar to that of intact virions. The vesicles were predominantly unilamellar and sealed, with both phosphatidyl ethanolamine and gangliosides symmetrically distributed in the bilayer. G protein was asymmetrically oriented, with about 80 percent accessible to exogenous protease. Addition of soybean phospholipid to the viral extract before dialysis resulted in vesicles that incorporated viral proteins and lipids quantitatively, but that were markedly decreased in buoyant density. The G neutralized protein-lipid vesicles were effective in eliciting specific anti-G antibodies that neutralized viral infectivity. Competitive radioimmunoassay showed that both reconstituted vesicles and a soluble form of G protein (Gs) were indistinguishable from purified VSV in their antibody binding properties. Addition of G protein-lipid vesicles of BHK-21 cells before, or simultaneously with, infection by VSV inhibited viral infectivity, as measured by two independent techniques (viral RNA production in the presence of actinomycin D and a neutral red assay of cell viability). The total inhibitory activity of G protein in the vesicular form was, however, less than 5 percent of that found for intact virus particles that have been inactivated by ultraviolet light irradiation. Gs was inactive as an inhibitor as determined by the RNA production assay.     

Identification and characterization of pseudorabies virus glycoprotein gM as a nonessential virion component.

Sequence analysis within BamHI fragment 3 of the pseudorabies virus (PrV) genome revealed an open reading frame homologous to the UL10 gene of herpes simplex virus. A rabbit antiserum directed against a synthetic oligopeptide representing the carboxy-terminal 18 amino acids of the predicted UL10 product recognized a major 45-kDa protein in lysates of purified Pr virions. In addition, a second protein of 90 kDa which could represent a dimeric form was observed. Enzymatic deglycosylation showed that the PrV UL10 protein is N glycosylated. Therefore, it was designated PrV gM according to its homolog in herpes simplex virus. A PrV mutant lacking ca. 60% of UL10 coding sequences was able to productively replicate on noncomplementing cells, demonstrating that PrV gM is not required for viral replication in cell culture. However, infectivity of the mutant virus was reduced and penetration was delayed, indicating a modulatory role of PrV gM in the initiation of infection.     

Protease of adenovirus type 2. Subcellular localization.

The subcellular localization of the adenovirus type 2 core polypeptide specific protease activity was investigated using an in vitro assay system. The protease activity was recovered exclusively from infected cell nuclei and was insoluble, sedimenting with the membrane fraction. Endogenous activity could be demonstrated in young virions which contain precursor PVII molecules. This protease activity only became sensitive to L-1-tosylamide-2-phenylethylchloromethyl ketone- or phenylmethylsulfonyl fluoride-mediated inhibition after disruption of the virus particles by sonication, suggesting that the enzyme was internally located. The putative precursors to virus particles, referred to as top components, which do not contain a full complement of viral DNA, did not contain protease activity. The protease released from sonicated virions converted exogenous PVII substrate molecules to polypeptide VII. The noninfectious H2ts1 virus particles synthesized at the nonpermissive temperature phenotypically resemble young virions, but unlike their wild type counterparts, were devoid of protease activity. The results show that the protease enters the precursor particles concurrently with the viral chromosome and that its presence is a prerequisite for the processing and subsequent maturation of infectious adenovirions.