Biochemical and biophysical properties of two strains of mosquito iridescent virus

Properties of “R” and “T” strains of mosquito iridescent virus (MIV) were investigated. Several differing biophysical properties of the two strains were observed due to size differences. The electrophoretic mobility curves of the two strains were somewhat different although the isoelectric points of pH 3.15 and pH 3.30 for RMIV and TMIV, respectively, were similar. The two strains appeared to be antigenically identical but the per cent protein, DNA, and lipid of the two were dissimilar. Other than size variation, no morphological differences between RMIV and TMIV could be detected.     

The DNA content of four small iridescent viruses: genome size, redundancy, and homology determined by renaturation kinetics

Fragmented DNA preparations from iridescent virus types 2, 6, 9, and 18 have been characterised for GC content and fragment size as a prelude to hybridisation studies. The genome size of the DNAs have been found to be 147 × 10⁶, 152 × 10⁶, 114 × 10⁶, and 114 × 10⁶ daltons for iridescent virus types 2, 6, 9, and 18, respectively, by studies of the kinetics of renaturation. Iridescent virus types 2 and 6 were found to contain a small proportion of DNA reannealing more rapidly than the bulk of the DNA, indicative of the presence of repeated sequences. In addition, the nucleotide sequence homology among the viruses has been investigated and found to range from 0 to 100%.     

Production and characterization of the cores of the "R" strain of mosquito iridescent virus.

Cores of “R” strain of mosquito iridescent virus (RMIV) were produced in vitro by reacting intact virus with chymotrypsin. Isolation of the cores from undegraded virus and outer capsid protein was accomplished by density gradient and differential centrifugations. Negatively stained core particles had a diameter of 176.1 ± 6.0 nm when examined in the electron microscope. The density of the particles as measured in cesium chloride gradients was 1.33, and the s_20,w was 3126 as measured in the analytical centrifuge. The molecular weight of the cores was calculated to be 1.84 × 10⁹ daltons. Protein, DNA, and lipid analysis of the cores accounted for all but 48.0% of the protein of intact virus particles. SDS-polyacrylamide gel electrophoresis of the cores compared with that of intact virus showed that only a 55,000 molecular-weight protein was absent in the former. The cores did not infect larvae or an Aedes aegypti cell line, and serological comparisons of intact virus and cores showed the two did not share common surface antigens.     

Characterization of the top component of the "T" strain of mosquito iridescent virus.

The molecular weight of the top component of TMIV was found to be 1.94 × 10⁹. The particles contain between 5.0 and 5.7% DNA, 4.3% lipid, and 87.1% protein. The antigenicity and electrophoretic mobility of top component differ from those of TMIV, although the number and molecular weight of top component proteins as determined in polyacrylamide gels appear identical to those of the virus. Top component particles are neither infectious nor do they affect the infectivity of virus suspensions.     

Structural characterization of the Molluscum contagiosum virus genome

The genome of Molluscum contagiosum virus (MCV) appears in an electron microscope as a linear, duplex DNA molecule having a mean contour length of 53.02 ± 1.87 μm. Vaccinia virus DNA, similarly processed and used for comparison, measures 53.03 ± 2.2 μm. The average molecular weight of MCV-DNA was calculated to be 118 × 10⁶. Single-stranded circles measuring twice the length of linear molecules were observed with both poxvirus DNAs following high levels of denaturation. However, partial denaturation profiles of MCV and vaccinia virus DNA differed markedly when mounted for microscopy under similar conditions. MCV-DNA consistently required an additional 10° equivalents to achieve comparable levels of denaturation. In contrast to vaccinia virus DNA, the most easily denaturable regions of the MCV genome appeared to be the ends of the molecule. When MCV-DNA was cleaved by restriction endonuclease BAM · HI, 12 fragments were produced ranging in size from 4 × 10⁵ to 31 × 10⁶ daltons as determined by agarose-gel electrophoresis and contour length measurements. Vaccinia virus DNA, similarly treated, produced more than 23 fragments which did not appear to correspond in size to those of MCV. With the aim of comparing the genetic and structural heterogeneity of the MCV genome, 11 independently isolated virus samples were analyzed by restriction endonuclease digestion and coelectrophoresis. Only three cleavage profiles were detected, with some fragments present in all three profiles, comigrating in agarose gels. We conclude, therefore, that the genome of Molluscum contagiosum virus is a continuous polynucleotide chain which is base paired to form a linear, duplex DNA molecule. Three genetic types of MCV were isolated from clinically typical skin lesions and identified in this study.     

Studies on virus-specific nucleic acids synthesized in vertebrate and mosquito cells infected with flaviviruses

Virus-specific RNA molecules synthesized in BHK 21 vertebrate cells and in Aedes albopictus mosquito cells infected with the flaviviruses Uganda S (US) or West Nile (WN) have been characterized. A single-stranded (ss) RNA of plus polarity sedimenting at about 42 S was present in the virus particles. 42 S plus strand RNA was also the predominant species of virus-specific ss RNA accumulating in infected cells of both vertebrate and insect origin. No similarity was detected between the large oligonucleotides generated by ribonuclease T₁ from WN virus and US virus-specific 42 S plus strand RNA, respectively. No poly(A) sequences are present in either the US virus or the WN virus-specific 42 S plus strand RNA molecules synthesized in BHK cells. The 42 S plus strand RNA molecules present in WN virus-infected BHK cells do contain, however, a “cap” structure m⁷GpppAmpN₁, and in some of these molecules, a further methyl group is introduced, giving rise to the “cap” structure m⁷GpppAmpN₁mpN₂. These “caps” are also present on the 42 S RNA of WN virus particles synthesized in BHK cells. In addition to the 42 S RNA, virus-specific ss RNA of low molecular weight (LMW-RNA) was detected in all virus-cell systems analyzed. A single species of LMW-RNA of 5 × 10⁴ daltons apparent molecular weight was present in US virus-infected vertebrate and insect cells. Two LMW-RNA species of about 6.5 × 10⁴ daltons (WM LMW-1 RNA) and 4.2 × 10⁴ daltons (WN LMW-2 RNA) molecular weight, respectively, were isolated from WN virus-infected BHK cells. Only the larger of these was detected in WN virus-infected insect cells. The LMW-1 RNA present in WN virus-infected BHK cells has been characterized in somewhat more detail. It contains virus-specific RNA sequences of plus strand polarity, is not “capped”, and does not contain a poly(A) sequence. None of the single-stranded, virus-specific RNA molecules synthesized in either vertebrate or mosquito cells bound to oligo(dT)-cellulose. Only a single species of virus-specific RNA containing minus strand sequences was detected in WN virus-infected BHK cells. This RNA was of genome size and was present as part of a double-stranded RNA complex containing 42 S RNA of both plus and minus polarity.     

Messenger Properties of TYMV-RNA

Turnip yellow mosaic virus (TYMV) RNA extracted from virions (“virion” TYMV-RNA) contains two types of molecules of molecular weight 2 × 10⁶: one which resists heat denaturation (“intact” TYMV-RNA) and one which yields smaller fragments upon heat denaturation (TYMV-RNA containing hidden breaks). A small RNA molecule of 0.2 × 10⁶ MW is also present in virions. “Virion” TYMV-RNA can therefore be separated into “intact” RNA and smaller molecules by heat denaturation followed by sucrose gradient centrifugation. The RNA from the sucrose gradient fractions were analyzed by formamide PAGE: The small RNA with a molecular weight of 0.2 × 10⁶ was separated from “intact” TYMV-RNA. The amount of coat protein synthesized by the RNA fractions when added to the wheat germ cell-free system was quite proportional to the amount of small RNA present. We conclude that coat protein synthesis is not directed by “intact” TYMV-RNA but by the small RNA. In addition, infectivity studies show that “intact” TYMV-RNA is fully infectious and thus it also contains the coat protein cistron.     

Sedimentation characteristics and molecular weights of three poxvirus DNAs.

DNA from vaccinia, fowlpox, and juncopox viruses was released from virions by gentle treatment with detergents and 2-mercaptoethanol or pronase and sedimented through neutral and alkaline sucrose gradients. Using T4 DNA with a S_0,w⁰ value of 61.4 as a standard, vaccinia DNA in neutral sucrose gradients had a S_0,w⁰ value of 62.1, whereas the sedimentation coefficients of fowlpox and juncopox DNAs were 72.0 and 69.3, respectively. In alkaline sucrose gradients, all three poxvirus DNAs showed an increased sedimentation rate compatible with a single-stranded circular molecule or a linear molecule twice the length of the native double-stranded molecule. This increased sedimentation rate in alkaline sucrose indicates that, like vaccinia, the DNA from the two avian poxviruses does not separate into complementary strands upon denaturation, but appears to contain covalent links between the complementary strands, located at or near the ends of the native DNA molecule.The sedimentation data of the native poxvirus DNA molecules was converted into molecular weights using a variety of expressions for relating the two parameters. Vaccinia DNA was seen to have a molecular weight of 125 ± 2 × 10⁶, whereas the fowlpox genome was 185 ± 5 x 10⁶. DNA from juncopox appeared to be slightly smaller than the fowlpox genome. The two avian poxviruses contain almost 50% more DNA than vaccinia, and are among the largest viral genomes known.     

Mouse adenovirus: growth of plaque-purified FL virus in cell lines and characterization of viral DNA

The FL strain of mouse adenovirus (AdFL) was plaque-purified and grown in mouse 3T6 cells. Purified virions resemble human adenoviruses by electron microscopy, and viral DNA shares physical properties with human adenovirus DNA. AdFL-DNA is a linear duplex with a molecular weight of 20 × 10⁶, shows evidence of protein covalently linked at each end, and forms single-strand circles after denaturation, indicating inverted repeat sequences at or near the ends of the molecule. However, less than 10% nucleotide sequence homology was found between AdFL-DNA and the DNAs of Ad2, 7, or 12. Also, the C + G content of AdFL-DNA (44%) is lower than that of human adenovirus DNAs. While there was reactivity between extracts of AdFL-infected cells and antiserum against human adenovirus, sera against the T antigens of human adenoviruses of groups A, B, and C did not react with AdFL-infected cell lysates. Six restriction endonucleases were used to cleave AdFL-DNA and to construct cleavage maps of the DNA. These maps differed from those of Ad2 or Ad5.     

Structure of frog virus 3 genome: size and arrangement of nucleotide sequences as determined by electron microscopy

The structural features of frog virus 3 (FV 3) DNA have been analyzed by electron microscopy. The FV 3 genome is a linear duplex molecule of 52.00 ± 5.60 μm in length which corresponds to 98.28 ± 10.58 X 106 daltons. To compare the structure of the genomes of FV 3, vaccinia virus, and herpes simplex virus, the three viral DNAs were denatured, quickly renatured, and examined in the electron microscope. The FV 3 DNA consisted of linear single strands while the vaccinia virus DNA contained reannealed duplexes and the herpesvirus DNA contained single-stranded circles with bushes or molecules in which two single-stranded circles were joined by a duplex region. These data suggest that the FV 3 genome contains neither crosslinked terminii nor inverted repeats. To construct a partial denaturation map of the FV 3 genome, the DNA was denatured with 85% formamide and examined in the electron microscope. The partially denatured molecules failed to match by their denatured regions when lined up end to end, suggesting that the molecules were dissimilar with respect to the arrangement of nucleotide sequences. However, a distinct denaturation map was obtained when the molecules were matched by their denatured regions rather than by the physical ends. The map revealed (1) a continuous stretch of about 40 μm (or 75.60 × 10⁶ daltons) of uniquely ordered sequences common to all molecules, and (2) a distribution of the ends within 20 to 28% of the map length. These observations suggest that FV 3 DNA molecules contain various circular permutations of a common overall nucleotide sequence and that the extent of circular permutation is restricted as in bacteriophage P22 (B. K. Tye, J. A. Huberman, and D. Botstein, 1974, J. Mot Biol. 85, 501–532).     

A study of the sequences of chicken embryo lethal orphan (CELO) virus DNA present in a transformed hamster cell line with use of specific fragments of the virus genome.

Chicken embryo lethal orphan (CELO) virus DNA (molecular weight 28.3 × 10⁶) was cleaved by the restriction enzyme EcoR1 into seven fragments whose molecular weights ranged from 1.8 × 10⁶ to 10.2 × 10⁶ as measured by gel electrophoresis and electron microscopy. ³²P-labeled CELO virus DNA was treated with EcoR1 and the resulting fragments were separated by gel electrophoresis. The kinetics of renaturation of the isolated fragments was measured in the presence of DNA extracted from a line of CELO virus-transformed hamster skin (THS) cells and from control hamster cells. THS cell DNA contained sequences homologous to only six of the seven EcoR1-produced CELO virus DNA fragments. No sequence homologous to a 2.0 × 10⁶ molecular weight fragment was detected indicating that the DNA of THS cells did not contain a complete copy of the virus genome.     

Search for specific DNAs in Creutzfeldt-Jakob infectious brain fractions using "nick translation"

Scrapie agent has been reported to contain an “essential DNA” component in the viroid size range (R. F. Marsh, T. B. Malone, J. S. Semancik, W. D. Lancaster, and R. P. Hanson, Nature (London) 275, 146–147, 1978). The human transmissible encephalopathy, Creutzfeldt-Jakob disease (CJ) was searched for unique CJ DNA components in highly infectious brain fractions containing negligible amounts of nuclear DNA. DNA was quantitatively extracted from these fractions and labeled to high specific activity by “nick translation” with ³²P-nucleotides under conditions where the labeled DNA maintained its original length. Labeled DNAs were analyzed by gel electrophoresis with special emphasis on molecules in the viroid size range. Reconstruction experiments showed that ?0.1 pg DNA could be detected with these procedures. No reproducible CJ specific DNA bands were detectable when compared to normal brain fractions. Restriction enzyme analysis of these DNAs also showed no CJ specific bands integrated into larger molecular weight DNA. Use of Brij 58 to release more infective physical particles into the rough endoplasmic reticulum also did not show CJ specific DNAs. Comparison of the amounts of known infective units by animal assay, assuming a 400-base DNA sequence, indicated that CJ specific DNAs should be detectable in these fractions with the methods used. These results appear to rule out usual forms of double-stranded or partially double-stranded DNAs as components of transmissible encephalopathy agents. A systematic analysis of other specific nucleic acid components of these agents is feasible using the highly sensitive in vitro labeling approach employed here.     

Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure.

DNA-binding proteins have been isolated from $\text{BHK21}\text{C13}$ cells infected with herpes simplex virus type 1 strain 17 syn⁺ by native and denatured DNA affinity chromatography. Sixteen proteins have been identified as specific to the infected cell on SDS polyacrylamide gradient gel electrophoresis and their molecular weights range from 145 × 10³ to 10.5 × 10³. After infection with herpes simplex virus many of the mock infected cell specific DNA binding proteins are no longer produced and this “switch off” of host cell protein synthesis is rapid. The virus-induced DNA-binding proteins fall into two quantitative classes and there is evidence for temporal control of their synthesis. One of the virus-induced DNA-binding proteins has a molecular weight identical to a polypeptide found in full but not empty particles and another binds preferentially to denatured DNA.     

Mechanism of infection by Epstein-Barr virus. II. Comparison of viral DNA from HR-1 and superinfected Raji cells by restriction enzymes.

Virus DNA (RS virus DNA) was directly isolated from Raji cells superinfected with Epstein-Barr virus derived from P3HR-1 cells and compared with original superinfecting virus DNA from P3HR-1 cells (HR-1 virus DNA) in agarose-gel electrophoresis after digestion with various restriction enzymes. EcoR-1 digestion of RS virus DNA produced 15 fragments identical to those from HR-1 virus DNA. However, two fragments, EcoR1 No. 6 (10 × 10⁶ daltons) and EcoR1 No. 11 (4.6 × 10⁶ daltons), observed in HR-1 virus DNA were not detected in RS virus DNA from superinfected Raji cells. In addition, the EcoR1 No. 4 (13.5 × 10⁶ daltons) fragment of RS virus DNA showed a molar ratio of 2 whereas HR-1 virus DNA produced the same fragment with a molar ratio of 1. Electrophoresis patterns of virus DNA digested with Hind III, Bam H-I, Hpa I, and Sal I were also examined. In general, both types of virus DNA produced similar patterns after gel electrophoresis, with minor differences in molar ratios after being treated with the restriction enzymes suggesting that RS virus DNA obtained by superinfection of Raji cells is basically identical to HR-1 virus DNA but may contain a population of DNA a little more heterogenous than HR-1 virus DNA.     

DNA from two Orgyia pseudotsugata baculoviruses: molecular weight determination by means of electron microscopy and restriction endonuclease analysis.

Both aqueous and urea-formamide procedures for spreading nucleic acid were employed for electron microscopic studies on the DNAs from the nucleopolyhedrosis bundle virus (NPBV) and the nucleopolyhedrosis single-rod virus (NPSV) both of which are pathogenic for Orgyia pseudotsugata. The molecular weight estimates via electron microscopy were derived by comparison of the mean length values for the double-stranded relaxed circular DNAs with that of SV40 DNA. The aqueous and urea-formamide spreading methods yielded NPSV DNA molecular weight values of 103 × 10⁶ and 104 × 10⁶ daltons, respectively, and molecular weight values of 87 × 10⁶ and 85 × 10⁶ daltons for NPBV DNA. These molecular weights were compared with molecular weight estimates from restriction endonuclease analysis, sedimentation analysis, and renaturation kinetic analysis. DNA located within the NPBV polyhedra but external to virions was characterized by restriction endonuclease analysis and examined by electron microscopy. It was determined to be composed of fragments of random size and to be viral origin.     

Analysis of the structure of the genome of pseudorabies virus.

Examination by electron microscopy of self-annealed single strands of pseudorabies virus DNA revealed the following structure: a sequence, (molecular weight, 9.9 × 10⁶) which is repeated in a complementary, inverted form on the other side of a short unique sequence (molecular weight, 6.0 × 10⁶), is present at one end of the molecule. The extreme end of the inverted repeat, the terminal region, is not repeated internally. A long unique sequence (molecular weight, 65 × 10⁶) comprises the remainder of the molecule. A sequence homologous to the end of the long unique sequence is also found approximately 350 bases from the end. In addition, examination of self-annealed exonuclease-treated DNA revealed the presence of double-stranded lariats, as well as smaller-than-unit-size circles, indicating that other sequences homologous to the end of the molecule are present at various distances from the ends. After exonuclease digestion of the ends and self-annealing, circular unit-size molecules were not observed. The number of nicks and gaps on individual pseudorabies viral DNA molecules varied between 1 and 10 per molecule; on the average, each molecule contained approximately 3.5 nicks or gaps. The nicks or gaps were found to be located at random sites along the molecule. No alkali-sensitive bonds were detected in the DNA.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

Adenovirus transcription: III. Mapping of viral RNA sequences in cells productively infected by adenovirus type 5

Separated strands of restriction endonuclease Eco R1 and Hpa 1 fragments of ³²P-labeled Adenovirus 5 DNA have been used in saturation hybridization experiments with cytoplasmic RNA extracted from human cells infected with Adenovirus type 5. The results of such experiments have allowed the construction of maps of the regions of the viral genome complementary to both “early” and “late” Adenovirus 5 RNA. At early times during the lytic cycle, about 25% of the viral genome is expressed as mRNA. Adenovirus 5 DNA sequences complementary to “early” mRNA comprise four discrete regions, two on each strand, of the viral genome: except in one instance, these correspond to the regions of the Adenovirus 2 genome complementary to “early” Adenovirus 2 mRNA. In the exceptional case, “early” Adenovirus 5 mRNA appears to contain not only sequences corresponding, at least in position, to those expressed in Adenovirus 2-infected cells, but also some additional, adjacent sequences, reminiscent of the situation in human cells infected with the Adenovirus 2/Simian virus 40 hybrid viruses, Ad2⁺ND₁ and Ad2⁺ND₃ (Flint et al., 1975a).All, or almost all, the information encoded by the Adenovirus 5 genome is expressed as mRNA during the lytic cycle and most of the exclusively “late” mRNA is complementary to the r strand of Adenovirus 5 DNA. The separated strands of fragments of ³²P-labeled Adenovirus 2 DNA generated by the restriction endonuclease Bam Hl have been used to improve the resolution of maps of both Adenovirus 2 and Adenovirus 5 “late” mRNA, which are described here.     

Analysis of human cancers,normal tissues,and verruce plantares for DNA sequences of human papillomavirus types 1 and 2

Comparatively little is known about human papillomaviruses (HPV) because they cannot be grown in tissue culture. We have in vitro labeled DNAs from two HPVs, HPV-1 which was isolated from plantar warts, and HPV-2 which was isolated from common hand warts, and used these DNAs to examine the homology between HPV-1 and HPV-2, to examine the state of the HPV genome in papillomavirus lesions, and to assay human cancer DNAs for HPV. The specific activities of the DNAs were 5.0 × 10⁷ to 1.1 × 10⁸ cpm/μg. The $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the HPV-1 and HPV-2 DNAs were 5 and 7 × 10^?4, respectively, consistent with a genome molecular weight of about 5.2 × 10⁶. Cross-hybridization of HPV-1 and HPV-2 DNAs revealed only 5–7% homology, confirming that these are distinct viruses. HPV-1 DNA was detected by Southern blot analysis in 9 of 10 plantar warts examined. No clear evidence was found for integrated viral sequences in DNAs from eight of the nine warts analyzed. Using these HPV-1 and HPV-2 probes, we have performed the first extensive and definitive molecular hybridization analysis of human cancer DNAs for HPV sequences. Human tumor DNAs were analyzed for HPV sequences by saturation hybridization using nick-translated HPV-1 and HPV-2 DNA probes. Reconstruction experiments with added HPV-1 or HPV-2 DNAs indicated that the probes could detect 0.1 copy of the viral genome per diploid equivalent of cellular DNA. No HPV-1 sequences were detected in DNAs from 156 human cancers (14 melanoma, 3 Ca skin, 5 Ca pharynx, 1 Ca esophagus, 4 Ca stomach, 5 Ca small intestine, 22 Ca colon, 14 Ca rectum, 25 squamous cell Ca lung, 3 adenocarcinoma lung, 4 oat cell Ca lung, 21 Ca kidney, 7 Ca bladder, 3 Ca ovary, 3 Ca cervix, 4 Ca prostate, 10 non-Hodgkin lymphoma, 2 reticulum cell sarcoma [spleen]), or 27 normal human tissues (1 skin, 10 tonsil, 8 colon, 8 kidney). No HPV-2 sequences were detected in DNAs from 145 human cancers (13 melanoma, 4 Ca skin, 2 Ca pharynx, 3 Ca mouth, 7 Ca esophagus, 4 Ca stomach, 3 Ca small intestine, 29 Ca colon, 15 Ca rectum, 25 Ca kidney, 15 Ca bladder, 2 Ca ovary, 6 Ca cervix, 4 Ca prostate, 2 Ca seminoma testes, 11 non-Hodgkin lymphoma) or 1 normal human ovary. These data are strong evidence that none of the cancer specimens assayed were induced by HPV-1 or HPV-2. However, additional work is required to fully evaluate whether HPVs are possible agents of human cancers, because the cancer types assayed in this study represent only about 50% of the cancer incidence in the United States, and because our probes would not detect sequences of other recognized HPV types (HPV-3, HPV-4, and HPV-5).     

Human fibroblasts transformed by the early region of SV40 DNA: analysis of "free" viral DNA sequences

Human fibroblastic cells (HF) were transformed with the early region of the simian virus 40 genome (0.15 – 0.73 map units) by using the DNA-calcium phosphate coprecipitation technique of F. L. Graham and A. J. Van der Eb (1973, Virology 52, 456–467). Transformation resulted in altered morphology and ability to grow in agarose. The SV40-transformed human fibroblasts (SVHF-A) have a limited life span and reach “senescence” after 10–11 passages. Analysis of the low molecular weight DNA extracted from SVHF-A cells shows a relatively high amount of free viral DNA sequences in circular supercoiled form. These circular molecules are very heterogeneous in size and contain sequences corresponding to the early region of the SV40 genome. Part of them may contain cellular DNA sequences as well. In situ hybridization experiments indicate that a minority of the SVHF-A cells (2–3%) are spontaneously induced to synthesize free viral DNA molecules and their frequency is increased by mitomycin C treatment. Immunofluorescence staining for SV40 T antigens also indicates that the cells producing free viral DNA contain higher T-antigen levels than the rest of the population. Our data suggest that the “free” viral DNA molecules derive from integrated viral sequences following replication in a minority of the cells rather than originating from a persistent extrachromosomal replication in every cell.