Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.     

Molecular cloning of biologically active proviral DNA of the anemia-inducing strain of spleen focus-forming virus.

Previously, we have molecularly cloned proviral DNA of a polycythemia-inducing strain of the spleen focus-forming virus (SFFVp). In this paper, we report that unintegrated proviral DNA of the anemia-inducing strain of SFFV (SFFVA) has been molecularly cloned into pBR322. This molecularly cloned DNA retains the biological activity of SFFVA, as infectious SFFV can be recovered from the DNA clone by marker rescue using a previously described two-stage cotransfection assay (Linemeyer et al., J. Virol. 35:710-721, 1980). The recovered SFFV retains an important property of the initial SFFVA which distinguishes SFFVA from SFFVP, namely, the ability of SFFVA to cause proliferation of erythroid cells in which hemoglobin synthesis is erythropoietin dependent. By utilizing a marker rescue technique, the splenomegaly and anemia characteristic of SFFVA-induced disease have been traced to a DNA fragment of SFFVA containing sequences coding for the env gene product. gp52. The results suggest that the differences in pathogenicity between SFFVP disease and SFFVA disease are an intrinsic property of the env gene products of these two variants of Friend virus, and future studies with the molecular clones of each strain should allow us to map regions of each env gene responsible for common and distinctive features of the erythroproliferative diseases induced by each virus.     

Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus.

Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.     

Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice.

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.     

Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat.

Rauscher and Friend spleen focus-forming viruses (R- and F-SFFVs) cause similar progressive erythroleukemias dependent upon a virus-encoded membrane glycoprotein. Moreover, these SFFV glycoproteins are immunologically related to each other and to the recombinant-type glycoproteins encoded by the env genes of dual tropic murine leukemia viruses. To better understand these diseases and the viral origins, we isolated a pathogenically active molecular clone of R-SFFV proviral DNA, sequenced its 3'-terminal 2,163-base-pair (bp) region, and compared these sequences with previously determined sequences of F-SFFV. The 516-bp R-SFFV long terminal repeat is highly homologous to those of F-SFFV and Friend murine leukemia virus, although only the latter contains a 65-bp direct repeat in its U3 region. The env gene of R-SFFV encodes a glycoprotein with 408 amino acids that is identical in its basic domain organization to the glycoprotein of F-SFFV. Thus, the junctions between the dual tropic-related and ecotropic sequences occur at the same nucleotide, and both SFFV env genes contain identical 585-bp deletions in their ecotropic domains and single-bp insertions which cause premature terminations at the same amino acid in their ecotropic p15E domains. Consistent with their independent origins, however, the env sequences of R- and F-SFFV are distinctive in both their 5' dual tropic-related and 3' ecotropic-related domains. Furthermore, there are several consistent amino acid differences between the polycythemic F-SFFV sequences and the anemia-inducing R-SFFV sequence. The striking similarities of the independently formed F- and R-SFFV env genes imply that all of the glycoprotein domains arranged in a precise organization may be required for its leukemogenic activity     

Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA.

The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication.     

Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector.

We studied the expression of a molecularly cloned human c-src gene, c-src-1, localized on chromosome 20, whose coding region consists of 11 exons and spans a 19.5-kilobase (kb) distance. Using a replication-competent retroviral vector derived from molecularly cloned Rous sarcoma virus DNA (pSRA-2), we obtained two constructs: one (pSR-CS) carrying the unmodified human c-src coding sequence and another (pSR-CVS) with a chimeric gene formed between the human c-src gene and the carboxy-terminal 12-amino acid v-src-specific coding sequence. From chicken embryo fibroblasts transfected with these DNA constructs, infectious viruses designated as WO CS and WO CVS, respectively, were recovered. WO CS virus did not cause cell transformation, whereas WO CVS induced cell transformation. Analyses of the proviral DNAs indicated that all introns were spliced out such that the 19-kb inserts were converted to 1.7-kb cDNA forms. Analyses of src proteins in infected cells, using monoclonal antibody MAb327 against v-src protein, showed the following results. The CVS and CS src proteins were about 60 and 61 kilodaltons in size, respectively; the specific protein kinase activity assayed in vitro of the CVS src protein was about 20-fold higher than that of the CS src protein and comparable to that of the v-src protein; the transforming CVS src protein reacted to an antibody against a v-src-specific peptide, whereas the CS src protein did not. These results indicate that the human c-src gene has a potential transforming ability and suggest that the v-src-specific sequence played an important role in the generation of Rous sarcoma virus.     

Specific neutralization of defective spleen focus-forming virus in Friend virus complex by rat antiserum.

The spleen focus-forming virus (SFFV), a rapidly transforming, replication-defective virus in Friend virus (FV) complex that is readily neutralized by antisera directed against its helper virus, was examined for the presence of SFFV-specific antigens. Antisera prepared in Fisher rats against an SFFV-infected Fisher rat embryo fibroblast line (SFFV-FRE) neutralized SFFV effectively, but not Friend-associated murine leukemia virus (F-MuLV) whether the latter was tested alone or was mixed with SFFV in the FV complex. In contrast, serum from mice immunized with SFFV-infected nonproducer mouse cells had little or no neutralizing activity against SFFV. Both absorption and immunoprecipitation studies indicate that the SFFV-specific antigen is immunologically related to xenotropic murine leukemia virus antigens. The role of both SFFV- and F-MuLV-specific antigens in the neutralization of SFFV suggests that this defective virus could be an antigenic mosaic and that viruses in the FV complex may participate in a undirectional form of phenotypic mixing.     

Structure of a cloned circular retroviral DNA containing a tRNA sequence between the terminal repeats.

In the course of analyzing a series of cloned circular retroviral DNAs, we recovered an unusual clone. The molecule consisted of a complete viral genome containing two copies of the long terminal repeat with extra sequences between the repeats. These extra bases proved to be a nearly complete DNA copy of a glycine tRNA, including bases that corresponded to modified and nonpairing bases of the mature tRNA. A model is proposed to account for the formation of the aberrant clone.     

Formation and structure of infectious DNA of spleen necrosis virus.

The kinetics of formation and the structure of infectious DNA of spleen necrosis virus were determined. Nonintegrated infectious viral DNA first appeared 18 to 24 h after infection of dividing cells and persisted for more than 14 days. The nonintegrated infectious viral DNA was in the form of either a double-stranded linear DNA with a molecular weight of 6 X 10(6), detected in both the cytoplasm and nucleus, or a closed circular DNA of the same molecular weight, detected primarily in the nucleus. Integrated infectious viral DNA appeared soon after the nonintegrated infectious viral DNA and was the predominant form of infectious viral DNA late after infection. Integration of the spleen necrosis virus DNA into the chicken cell genome was demonstrated by three independent criteria. Nucleic acid hybridization indicated that the linear infectious viral DNA had a 5- to 10-fold higher specific infectivity than either the closed circular or integrated infectious viral DNA. Infectious viral DNA did not appear in infected stationary cells, indicating some cellular influence on the formation of infectious viral DNA.     

Rescue of endogenous 30S retroviral sequences from mouse cells by baboon type C virus.

Mus musculus SC-1 cells were infected with M7 baboon type C virus. The progeny of this infection included viral pseudotypes that contained M7 helper virus and endogenous 30S retrovirus-associated sequences derived from SC-1 cells (RAS). The RAS sequences are unrelated by nucleic acid hybridization criteria to previously described types of murine retroviruses and do not code for known murine viral structural proteins. The RAS genome is present in multiple copies in the DNA of laboratory (M. musculus) and Asian (M. caroli and M. cervicolor) mice, is expressed in the RNA of uninfected mouse cells, and can be efficiently rescued by type C, but not type B, viruses. RAS is closely related to 30S virus-associated RNA in NIH/3T3 and BALB/c JLSV-9 cells and may be analogous to the defective 30S RNA sequences found in rats.     

Friend strain of spleen focus-forming virus: a recombinant between mouse type C ecotropic viral sequences and sequences related to xenotropic virus.

The genome of the Friend strain of the spleen focus-forming virus (SFFV) has been analyzed by molecular hybridization. SFFV is composed of genetic sequences homologous to Friend type C helper virus (F-MuLV) and SFFV-specific sequences not present in F-MuLV. These SFFV-specific sequences are present in both the Friend and Rauscher strains of murine erythroleukemia virus. The SFFV-specific sequences are partially homologous to three separate strains of mouse xenotropic virus but not to several cloned mouse ecotropic viruses. Thus, the Friend strain of SFFV appears to be a recombinant between a portion of the F-MuLV genome and RNA sequences that are highly related to murine xenotropic viruses. The implications of the acquisition of the xenotropic virus-related sequences are discussed in relation to the leukemogenicity of SFFV, and a model for the pathogenicity of other murine leukemia-inducing viruses is proposed.     

Rapid purification of biologically active individual histone messenger RNAs by hybridization to cloned DNA linked to cellulose.

We describe a rapid and simple method for the purification of biologically active messenger RNAs. The method allows the isolation in a few hours of specific mRNAs from either whole cell or polysomal RNA even if the RNA represents less than 1% of the starting molecules. We used, as a model, cloned sea urchin (Strongylocentrotus purpuratus) histone gene fragments linked to cellulose by the method of B. E. Noyes & G. R. Stark (1975) Cell 5, 301--310) as hybridization probes to isolate specific histone mRNAs from whole cell and polysomal RNA extracts. RNAs isolated in this manner maintain their biological activity, serving as templates for histone proteins in a wheat-germ, cell-free protein translation system. In addition, radiolabeled histone-specific RNA purified from cleavage stage sea urchin embryos, pulse labeled for short periods of time and analyzed on denaturing polyacrylamide gels, was the same size as mature histone mRNA's.     

Dimethyl sulfoxide affects the amount of extrachromosomal spleen focus-forming virus DNA in murine erythroleukemia cells.

We used Southern blot hybridization to titrate and map restriction enzyme cleavage sites of a 6.3-kilobase-pair species of extrachromosomal viral DNA found in derivatives of the 745A line of murine erythroleukemia cells, which vary in their ability to be induced to differentiate by dimethyl sulfoxide (DMSO). Greater than an eightfold variation was observed in the amount of this DNA, with the largest amounts being found in cells that were resistant to the induction of differentiation by DMSO. This increase in the level of extrachromosomal viral DNA was found to be dependent upon the continued presence of DMSO in the culture medium. The increase was shown not to be due to an immediate stimulatory effect of this agent on the synthesis or maintenance of this DNA, since cell lines sensitive to the differentiation-inducing effects of DMSO were shown to undergo a transient reduction in the amount of extrachromosomal viral DNA after the addition of DMSO to the culture medium. In addition to the 6.3-kilobase-pair linear form found in the cytoplasm, in some preparations two hybridizing bands were observed that migrated in agarose gels in the position expected of covalently closed circular species of viral DNA. Restriction enzyme mapping of the cytoplasmic linear form indicated a close relationship of this DNA to two polycythemic strains of spleen focus-forming virus that have been molecularly cloned by other workers. No obvious change in the number or arrangement of chromosomal viral sequences could be detected after treating cells with DMSO. Thus, the exposure of murine erythroleukemia cells to DMSO caused an obvious change in the amount of extrachromosomal spleen focus-forming virus DNA but no obvious change in the integration of the provirus.     

Comparison of infectious JC virus DNAs cloned from human brain.

We cloned JC virus DNA obtained directly from brain tissue of 10 cases of progressive multifocal leukoencephalopathy and compared DNAs by restriction endonuclease mapping. Before cloning, each DNA preparation was homogeneous with respect to restriction patterns, but with the cloned DNAs we found variability in three regions of the genome among DNAs from different cases. There was a region of hypervariability between 0.67 and 0.725 map units; no two DNAs were exactly alike in this region. We determined that the origin of DNA replication also was in this region at 0.69 +/- 0.02 map units. In 4 of the 10 DNAs examined there was a deletion of approximately 75 base pairs between 0.14 and 0.235 map units, the region presumed to contain the codons for the C-terminal ends of the structural protein Vpl and for T antigen. JC virus DNA from these same four cases had an additional HincII-HpaI site at 0.895 map units in the presumptive Vp3 and Vp2 coding regions. Overall, no two JC virus genomes were identical although all were from fatal central nervous system infections and were infectious in vitro. Our restriction patterns suggest that there are two subtypes of JC virus circulating in the population.     

Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac.

Infection of macaque monkeys with simian immunodeficiency virus (SIV) is probably the best animal model currently available for studying acquired immunodeficiency syndrome. In this report, we describe three infectious molecular clones of SIVmac and one of human immunodeficiency virus type 2 (HIV-2) and their use in the study of cell and species specificity, animal infection, and the relationship of gene sequence to function. Replication of the cloned viruses in different cell lines varied dramatically. Some human CD4+ cell lines (HUT 78 and MT-4) supported the replication of SIVmac and HIV-2, while others (CEM and Jurkat-T) supported the replication of HIV-2 but not SIVmac. Growth of cloned virus in macaque lymphocytes in vitro was predictive of macaque infection in vivo. Macaque lymphocytes supported the replication of SIVmac239 and SIVmac251 but not SIVmac142 or HIV-2ROD. Using virus recovery and antibody response as criteria for infection, macaques that received cloned SIVmac251 and SIVmac239 became infected, while macaques receiving cloned SIVmac142 and HIV-2ROD did not become infected. Nucleotide sequences from the envelope region of all four cloned viruses demonstrated that there is considerable flexibility in the location of the translational termination (stop) signal. These infectious molecular clones will be very useful for future studies directed at the molecular basis for persistence, pathogenicity, tropism, and cell and species specificity.     

Isolation of infectious xenotropic mouse type C virus by transfection of a heterologous cell with DNA from a transformed mouse cell

An endogenous xenotropic type C virus has been isolated from a Kirsten sarcoma virus-transformed BALB/c mouse cell line by transfection of a mink fibroblast cell with the DNA from the transformed cells. The results indicate that transfection may be used as a technique to isolate this endogenous type C virus without the need to chemically induce the cell line containing the provirus prior to attempting to isolate the virus.     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

Roles of helper and defective retroviral genomes in murine erythroleukemia: studies of spleen focus-forming virus in the absence of helper.

Retroviruses that cause acute oncogenesis are generally complexes of a replication-competent helper virus and a replication-defective component. However, the pure defective components have not been previously available. We prepared the defective spleen focus-forming virus component of Rauscher erythroleukemia virus (R-SFFV) by transfecting a colinear R-SFFV DNA clone into a retroviral packaging cell line (psi 2 cells). The transfected cells released virus (psi 2/SFFV) that was free of helper virus and that induced erythropoietin-dependent erythroid burst formation in bone marrow cultures. When injected into normal adult NIH/Swiss mice in moderate doses, psi 2/SFFV caused a rapid splenic erythroblastosis that regressed. Extensive erythroblastosis could be maintained by repeated injections of psi 2/SFFV into anemic mice or by the addition of a helper virus. We conclude that R-SFFV alone causes proliferation but not immortalization of a population of erythroblasts that is normally replenished from a precursor stem cell pool. Because these precursor cells are inefficiently infected, a single moderate inoculum of psi 2/SFFV causes a wave of erythroblastosis. The properties of the proliferating erythroblasts are substantially determined by the R-SFFV viral component.     

Crystallization of biologically active hemagglutinin-neuraminidase glycoprotein dimers proteolytically cleaved from human parainfluenza virus type 1.

We isolated, purified, and characterized the hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 1, with the ultimate goal of producing crystals suitable for three-dimensional X-ray structure analysis. Pronase was used to cleave the globular head of the HN molecule directly from virus particles, forming HN monomers and dimers. The purified dimers retained neuraminidase and hemadsorption activity and were recognized by 14 anti-HN monoclonal antibodies, demonstrating intact HN antigenic structure and function. N-terminal sequence analysis of the dimers showed that cleavage had occurred at amino acid 136 or 137, freeing the C-terminal 438 or 439 amino acids. On electron micrography, the dimer appeared as two box-shaped structures, each approximately 5 by 5 nm. When the purified HN dimers were crystallized in hanging drops by vapor diffusion against 20% polyethylene glycol 3350, they formed both rectangular plates and needlelike crystals. The rectangular crystals diffracted X-rays, indicating an ordered atomic structure. However, the resolution was approximately 10 A (1 nm), insufficient for three-dimensional structural analysis. Experiments to improve the resolution by increasing the size and quality of the crystals are in progress.