Nucleic acid molecules: new microdiffusion technique for visualization.

A microdiffusion technique, developed for visualization of nucleic acid molecules in the electron microscope, requires less than 0.01 microgram of nucleic acid. Although originally developed for free nucleic acids, the method can be applied to virion suspensions for direct visualization of their genomes; less than 10(10) virions per milliliter are required. Results agree well with those yielded by the diffusion technique of Lang, Kleinschmidt, and Zahn.

Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions.

Based on physical and chemical determinations, the mnolecular weight of the type 4 adeno-satellite virus is 5.4 X 10(6) daltons, and the virion contains 1.4 X 10(6) daltons of DNA. Denaturation and renaturation studies indicate that the viral genome is a single-stranded DNA molecule and that each virion contains either a minus or a plus strand. Upon extraction, the minus and plus strands unite to form double-stranded DNA molecules with no obvious excess of unpaired strands.

Nonencapsidated infectious DNA of adeno-satellite virus in cells coinfected with herpesvirus.

Adeno-associated satellite viruses produce antigen detectable by immunofluorescence but not infectious virus in tissue culture cells coinfected with herpes simplex virus. Analysis of DNA extracts from these infected cells shows that large amounts of infectious satellite virus DNA are produced but not encapsidated in the system. This result indicates that satellite virus may be defective at the maturation step.

Evolution of a defective virus from a cellular defense mechanism.

Adeno-associated virus is a defective DNA virus, requiring the presence of a helper virus in order to replicate. In this paper we consider its origin in light of several observations, most notably the following: its own replication inhibits that of the helper virus; its DNA structure resembles that of transposable (moveable) elements; and extrachromosomal circles of DNA, about the size of adeno-associated virus DNA, have been found recently in eukaryotic cells. We have arrived at a hypothesis consisting of two main ideas: (1) that cells may use transposable DNA as a mechanism of defense against viral attack, and (2) that adeno-associated virus may have evolved directly from this cellular defense mechanism.

Defective parvoviruses acquired via the transplacental route protect mice against lethal adenovirus infection.

Adeno-associated virus type 1 (AAV-1) interfered with the replication of its murine adenovirus (MAV) helper in primary mouse kidney cells and in 1-day-old ICR mice. Mice carrying AAV-1 acquired via the transplacental route were protected against lethal infection with MAV. The replication of AAV-1 in these mice could be triggered by multiple challenges with MAV, and antibodies to AAV-1 were subsequently detected.

Inhibition of the replication of parvovirus X14 by 5-iodo-2'-deoxyuridine pre-treatment of cell cultures.

Pre-treatment of rat embryo cell cultures with 5-iodo-2'-deoxyuridine (IdUrd) inhibits the replication of parvovirus X14. Reduced yields of haemagglutinating and infectious particles were observed. Adsorption of virus to cells was not affected, but both virus protein and DNA synthesis were inhibited. Fewer cells were capable of supporting protein or antigen synthesis as determined by immunofluorescence. Virus-specific DNA was detected in IdUrd pre-treated cells, but the amount synthesized was considerably less than that from control cultures. Cellular DNA synthesis was also inhibited in IdUrd pre-treated cells. Therefore, the replication of parvoviruses appears dependent upon host cell factors involved in cellular DNA synthesis.

Isoelectric focusing of parvoviruses.

Adenovirus-associated virus type 4 and X14 migrate during electrophoresis to pH 2.6 in sucrose-stabilized, pH 2.5-6.0 gradients. Naturally occurring empty capsids appear to have the same isoelectric point as complete virus particles.

Properties of adeno-associated virus (type 1) replicated in rodent cells by murine adenovirus.

We report for the first time the replication of infectious adeno-associated virus type 1 (AAV-1) in rodent cells [primary mouse kidney (PMK) and mouse L929 cells] using murine adenovirus (MAV) as a helper virus and also the production of AAV-I virus antigen by herpes simplex virus type I (HSV-I) with its temperature-sensitive mutant ts 200 in mouse neuroblastoma (NB) cells. The infectious AAV virions produced by MAV on L cells had a buoyant density of 1.41 2ml in caesium chloride gradients.

Transplacental infection with adeno-associated virus type 1 in mice.

Adeno-associated type 1 parvovirus (AAV) was detected in the kidneys and lungs of fetuses and newborns, when pregnant mice were injected subcutaneously with AAV type 1 and murine adenovirus as a helper virus. These findings clearly indicate that transplacental infection with AAV in rodents has been achieved.

Comparative study of seven picornaviruses of man.

Jamison, Richard M. (Baylor University College of Medicine, Houston, Tex.), and Heather D. Mayor. Comparative study of seven picornaviruses of man. J. Bacteriol. 91:1971-1976. 1966.-The buoyant densities in CsCl and potassium tartrate of seven acid-resistant picornaviruses of man were determined. The viruses studied were found to be a relatively homogeneous group. Buoyant densities ranged from 1.32 to 1.35 g/cc in CsCl and 1.21 to 1.23 g/cc in potassium tartrate. The buoyant density of each virus did not vary by more than 0.01 g/cc. The host cell of origin and passage level did not influence the buoyant density of the virions. However, measurements of the diameters of purified virions by a standard method have shown that there are considerable size variations among these picornaviruses. The diameters ranged from 18 to 25 mmu. Echovirus 19 and poliovirus 1 were significantly larger (mean diameter, 23.5 mmu) than echoviruses 4, 6, 16, and 23, and coxsackievirus B2 (mean diameter, 21.0 mmu).

Characterization of heavy particles of adeno-associated virus type 1.

The temperature-sensitive mutant ts4 of adenovirus type 2 (Ad-2) is capable of complementing adeno-associated virus type 1 (AAV-1) in HEp2, KB and HEK cells at 34 degrees C and 39 degrees C when used as a helper virus. Heavy non-infectious AAV-1 particles can be generated by using the mutant ts4 in HEp2 cells. When AAV-1 is grown in serial passages in HEp2 cells, both the wild-type Ad-2 and the mutant ts4 give rise to heavy, less infectious AAV-1 particles. The heavy AAV-1 particles generated by Ad-2 in advanced serial passages retain the property of having CF and IF antigens, but the AAV-1 generated by the mutant in advanced serial passages lose this property. There is no appreciable difference in the particle counts made by electron microscopy of AAV-1 preparations generated either by Ad-2 or the mutant ts4. Analysis by polyacrylamide gel electrophoresis of purified heavy AAV generated by ts4 indicates that in late passage an additional polypeptide of higher mol. wt. than the three structural polypeptides is detected.

Interference between two adeno-associated satellite viruses: a three-component system.

Adenovirus-associated satellite viruses interfere with the replication of their helper adenoviruses. According to a previous report, this interference is not mediated by interferon. A three-component system comprising simian adenovirus SV15 and satellites types 1 and 4 was studied to determine whether satellite viruses also interfere with one another. Satellite type 1 interfered with the replication of type 4 and vice versa. The degree of interference was directly proportional to the dose of interfering satellite. The events leading to mutual satellite interference were operative during the first 12 hr of replication, the period associated with active synthesis of viral deoxyribonucleic acid.

Biophysical and biochemical studies on rhinovirus and poliovirus. II. Chemical and hydrodynamic analysis of the rhinovirion.

Chemical analysis of rhinovirus 14 revealed a ribonucleic acid (RNA) content of 29.8% and a high adenylic acid content (35%). A partial specific volume of 0.682 cm(3)/g was obtained for the rhinovirion. Rhinovirus and poliovirus had identical sedimentation coefficients of 158S. A diffusion coefficient of 1.71 x 10(-7) cm(2)/sec was consistent with a hydrated diameter of 25 nm for the rhinovirion. The calculated molecular weights of the rhinovirion and its genome were 7.1 x 10(6) and 2.1 x 10(6) daltons, respectively. Sedimentation analysis of infectious RNA confirmed the similarity of the molecular size of the poliovirus and rhinovirus genomes.

The evolution of defective and autonomous parvoviruses.

Because of the small size and genetic simplicity of small DNA viruses, parvoviruses would appear to be excellent models for studying viral evolution and adaptation. In an earlier publication we hypothesized the evolution of sequences of cellular "junk" DNA into protective interfering transposons. These transposons would interfere with invading pathogenic viruses by competing with the pathogen DNA for replicative enzymes. We speculated that a small, defective parvovirus, the adeno-associated virus (AAV), which usually requires the presence of a pathogenic helper virus to replicate, may have evolved from such a piece of cellular "junk" DNA. Our theory predicted that AAVs, as a consequence of their defective nature, developed under pressures favoring maintenance of their transposon like qualities. In contrast, disease-causing, autonomous, non-defective parvoviruses such as the B19 agent of humans and the canine parvovirus, even though their origins may have been in cellular DNA, would appear to have developed under totally different evolutionary pressures. In this paper we will present evidence for a common ancestry for the defective and autonomous parvoviruses and discuss the divergent paths this evolution may have taken in establishing the two genera.

The replication of rodent parvovirus X14.

The replication of rodent parvovirus X14 DNA has been studied in rat embryo tissue culture cells. Virus DNA was isolated from I M-NaCl-SDS-pronase supernatant fluids from 24 h after infection. The majority of this DNA was 1-7 mum in length and double-stranded, indicating that it was an intermediate in the replication cycle of this single-stranded DNA virus. Single-stranded DNA of equivalent length was isolated directly from X14 virions. The buoyant density of this DNA was 1-728 g/ml whereas the double-stranded form banded at 1-714 g/ml in caesium chloride gradients. Difficulties in detecting significant amounts of single-stranded viral DNA directly from infected cells would appear to indicate that progeny single-stranded DNA is rapidly encapsidated after synthesis.

Deoxyribonucleic acid of adeno-associated satellite virus.

Staining patterns suggest that in the adeno-associated satellite virion there exist quasi single-stranded regions which are renatured after extraction to exhibit double strandedness.