Infection Of NIH 3T3 Cells Research Articles

Cloning of adenovirus type 12 E1 genes into a retroviral vector and their differential splicing in mouse cells

Early region 1 ( E1) of adenovirus type 12 (Ad12) genome is able to transform nonpermissive primary rodent cells in vivo and in vitro. To analyse the role of the E1a gene products alone or in connection with the 58-kDa protein encoded by E1b during oncogenic transformation, we have cloned genomic fragments of both subregions into the retroviral vector, pZIP-NeoSV(X)1. Both constructs are expressed in mouse 3T3 cells, but, in contrast to E1b, the amount of genomic retroviral RNA carrying E1a-specific sequences was low in transfected psi2 cells and not detectable in infected NIH3T3 cells. Nevertheless, we could demonstrate the integration of the complete E1a-carrying protovirus into the NIH3T3 genome. However, after infection of primary mouse embryo fibroblasts, high retrovirus-mediated expression of E1a leads to the immortalization of these cells. In the derived cell line, only the 13S transcript and the unspliced form of E1a RNA could be demonstrated, but not the 12S transcript. These results demonstrate that the ration of genomic vs. subgenomic retroviral RNAs of Ad12 E1-carrying is dependent on the cloned insert and the cell system used.

Chromatin structure of recombinant Moloney murine leukemia virus proviral DNAs that contain tax-responsive sequences from human T-cell lymphotropic virus type II in the presence and absence of tax.

Human T-cell lymphotropic virus types I and II (HTLV-I and HTLV-II) are replication-competent retroviruses which contain two additional regulatory proteins, tax and rex. tax is a transcriptional transactivator of the HTLV-I or HTLV-II long terminal repeat (LTR) and also of some heterologous promoters. To investigate the mechanism of tax transactivation, we used chimeric Moloney murine leukemia viruses (M-MuLVs) with LTRs containing tax-responsive sequences from the HTLV-II LTR (nucleotides -273 to -32). Mo+HTLV-II+ M-MuLV contained the HTLV II sequences inserted into the wild-type M-MuLV LTR at nucleotide -150, whereas delta Mo+HTLV-II+ M-MuLV contained the same sequences inserted into an M-MuLV LTR lacking its own enhancer region. HTLV-II tax (tax II)-positive mouse cells (15S-5a) infected with Mo+HTLV-II+ M-MuLV or delta Mo+HTLV-II+ M-MuLV showed higher rates of viral transcription in nuclear run-on assays than did infected tax-negative NIH 3T3 cells. The chromatin structure of these viruses was investigated by high-resolution mapping of DNase I-hypersensitive (HS) sites. Three prominent HS sites were associated with HTLV-II sequences in proviral chromatin both in tax-positive and in tax-negative cells. The spacing resembled that of the 21-base-pair (bp) repeats, but the HS sites were displaced approximately 50 bp upstream of the 21-bp repeats. This suggested that cellular proteins bound to the HTLV-II sequences in the presence or absence of tax. No direct effect of tax on chromatin structure was found. These in vivo results were consistent with results of in vitro DNase footprinting studies performed by other investigators.

Insertional mutagenesis of the murine cytomegalovirus genome: One prominent α gene (ie2) is dispensable for growth

We have utilized insertional mutagenesis to investigate the functional importance of murine cytomegalovirus (MCMV) immediate early (IE or α) region 2 (ie2) in replication. We constructed a recombinant virus, RM408, that carries the Escherichia coli lacZ gene under transcriptional control of the MCMV α promoter/enhancer inserted within the ie2 transcription unit. RM408 carries the first defined genetic mutation in a specific CMV gene. After infection of NIH3T3 cells, RM408 expressed β-galactosidase in abundance and with kinetics indistinguishable from those of the natural ie1 gene product which is left intact in the recombinant. We detected no expression from the ie2 region in RM408-infected cells. Growth kinetics, yield, and expression of other viral genes in RM408 was similar to wild-type MCMV, indicating that the ie2 gene product was nonessential for growth in cell culture.

Analysis of a transformed cell line using antisense c- fos RNA

Simian sarcoma virus (SSV)-infected NIH-3T3 cells (SSV-NIH-3T3), express a homologue of platelet-derived growth factor, (PDGF) a powerful inducer of the c- fos gene. We have used these cells to test the hypothesis that autocrine stimulation by PDGF-like molecules leads to c- fos expression which is functional in the transformed phenotype. We have transfected SSV-NIH-3T3 cells with a c- fos antisense-RNA expression vector, pSV sof, or control plasmids. pSV sof-transfected cells exhibit markedly decreased c- fos mRNA and protein levels, restored density-dependent growth arrest and reduced (three of five clones) tumorigenicity compared to control lines. The results confirm that c- fos cooperates in the transformed phenotype of SSV-NIH-3T3 cells.

Use of retroviral vectors for mapping of splice sites in cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) genomic sequences coding for virus early functions were introduced into a retroviral vector in order to produce cDNAs of the viral early region. Two constructs differing in the length of control sequences preceding the E6 open reading frame were transfected into Psi-2 cells and the released retroviral stock was used to infect NIH3T3 cells. The proviral sequences were rescued from antibiotic G418-resistant virus-infected cells after fusion with Cos cells, amplified as plasmids in Escherichia coli and analysed. Nucleotide sequencing showed that the splicing signals used in the construct containing only the early coding region are the same as in CRPV-expressing tumours, linking the beginning of E1 to the middle of E2. On the other hand, in a construct including most of the long control region a splice donor site located in the 5' end of this region, at position 7810, was very efficiently used, totally excluding the use of the donor site at position 1371. None of the constructs containing CRPV sequences transcribed from Moloney murine leukaemia virus promoter was able to transform mouse fibroblasts after DNA transfection.

Oncogenes modulate cellular gene expression and repress glucocorticoid regulated gene transcription

The v-mos oncogene was subjected to the transcriptional control of the MMTV LTR and introduced by transfection into NIH 3T3 cells. The LTR v-mos gene was induced by the addition of glucocorticoid hormone to the growth medium of cells synchronized by culturing in 1.5% FCS for 36 h. The effects of p37 v-mos expression were monitored. The endogenous c-myc gene is induced as a consequence of p37 v-mos expression in a transient fashion, reaching a maximum of expression after 8 h. Induction of the c-myc gene was observed at the level of its transcriptional rate and at the level of mRNA concentration. Omithine decarboxylase (ODC) mRNA was induced constitutively and high levels were found 8 and 25 h after v-mos induction. H4 histone mRNA is elevated at 25 h after hormone addition at a time when the mitogenic stimulus of v-mos causes DNA synthesis. The expression of actin mRNA is not affected by the v-mos oncogene. We have previously described a modulation of glucocorticoid dependent gene expression by oncogenes. In an extension of these observations the consequences of expression of the v-mos and the v-ras oncogenes were also studied in retrovirally infected NIH 3T3 cells. MMTV LTR constructs transfected into the infected cells could only be transiently induced by glucocorticoid hormone. The presence of the p37 v-mos and the p21 v-ras oncoproteins causes a repression of glucocorticoid hormone dependent gene transcription.

Human cytomegalovirus (HCMV) enhances bovine papilloma virus (BPV) transformation in vitro.

Infection of NIH 3T3 cells with a combination of HCMV and BPV resulted in more foci than infection with BPV alone. Foci were microscopically apparent at 4 days in the mixed infection and did not appear until 2 days later in the cultures infected with BPV alone. The enhancement was abolished by heat inactivation of the HCMV and also when the HCMV was replaced by a "mock inoculum." Southern blot analysis of cellular DNA from transformed cells showed a similar amount of extrachromosomal BPV DNA in cells infected by BPV alone and in cells co-infected with HCMV. No HCMV antigens could be found in these cells by immunofluorescence. The mechanisms of the enhancement are not known. Stimulation of host DNA synthesis by HCMV could possibly increase the transforming efficiency of BPV. Alternatively, the increase in BPV transforming efficiency could be due to a transient increase in BPV-1 transcription by an HCMV transcriptional transactivation factor. Since both HCMV and human papillomaviruses are commonly found in the uterine cervix, HCMV may play a role in human cervical cancer by enhancing the carcinogenic potential of human papillomavirus.

Specific sequences of the env gene determine the host range of two XC-negative viruses of the rauscher virus complex

Two viruses which do not give rise to XC plaques in the standard XC assay (XC-negative) have been isolated from the Rauscher virus (RV) complex. These viruses differ in their host range. One, R-MCF-1, is dualtropic and will therefore infect both murine and non-murine cells. However, unlike other mink cell focus-inducing (MCF) viruses, it cannot infect NIH 3T3 cells. The other, R-XC −, is ecotropic. It will infect murine cells, including NIH 3T3 cells, but does not infect mink lung cells. Analysis of hybrid viruses, in which homologous regions of the genomes of R-MCF-1 and R-XC − virus were exchanged, indicated that the NH 2-terminal portion of the gp70 is responsible for the particular host ranges of these viruses. The nucleotide sequence of the env gene of R-XC − virus was therefore determined and compared with the known env sequences of ecotropic MLVs and dualtropic MCF viruses of the Rauscher and Friend virus complexes. R-XC − virus was found to be a recombinant virus. Its env gene contained sequences derived from an endogenous env gene which were closely related to those of the MCF viruses but differed from any previously described sequences. The particular properties of R-MCF-1 and R-XC − virus suggest that the two viruses arose by recombination between R-MLV and two endogenous env sequences which differ from those of the known MCF viruses. If so, this suggests that the mouse genome contains at least five env sequences which can give rise to MCF-like viruses. In addition, since the host range and interference properties of R-XC − virus are very similar to those of the previously described ecotropic recombinant viruses (4), it may be that the ecotropic recombinant viruses arose by recombination with the same endogenous env sequences as did R-XC − virus.

Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.

A retrovirus-derived vector called self-inactivating (SIN) vector was designed for the transduction of whole genes into mammalian cells. SIN vectors contain a deletion of 299 base pairs in the 3' long terminal repeat (LTR), which includes sequences encoding the enhancer and promoter functions. When viruses derived from such vectors were used to infect NIH 3T3 cells, the deletion was transferred to the 5' LTR, resulting in the transcriptional inactivation of the provirus in the infected cell. Introduction of a hybrid gene (human metallothionein-promoted c-fos) into cells via a SIN vector was not associated with rearrangements and led to the formation of an authentic mRNA transcript, which in some cases was induced by cadmium. SIN vectors should be particularly useful in gene transfer experiments designed to study the regulated expression of genes in mammalian cells. Absence of enhancer and promoter sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular oncogenes and may provide a safer alternative to be used in human gene therapy.

Molecular basis of alpha 1-antitrypsin deficiency and its potential therapy by gene transfer.

The gene for alpha 1-antitrypsin, a serum anti-protease, has been cloned and sequenced. The underlying mutation in the PiZ allele has been identified as a G to A conversion giving rise to the substitution of glu by lys at position 342. Preparation of specific probes has allowed prenatal diagnosis. Recombinant retroviruses containing the normal human alpha 1-antitrypsin gene have been constructed and used to infect NIH3T3 cells. Analysis of DNA, RNA and protein indicate that successful incorporation of the alpha 1-antitrypsin was achieved and that the gene was capable of being expressed. The feasibility of genetic replacement therapy has been demonstrated and further experiments justified.

Recognition of mos-related proteins with an antiserum to a peptide of the v-mos gene product.

A mos-specific antiserum was generated by injection of rabbits with a peptide predicted from the sequence of the v-mos gene of Moloney murine sarcoma virus (MuSV) strain 124. The peptide is composed of amino acids 37-55 (cyclized at the cysteine residues) conjugated to keyhole limpet haemocyanin. This serum [anti-mos(37-55c)] specifically recognized p37mos in MuSV-124 acutely infected NIH-3T3 cells, P85gag-mos in 6m2 cells, an NRK clone infected with the temperature-sensitive mutant (ts110) of Moloney MuSV, and P100gag-mos in 54-5A4 cells, an NRK clone infected with a spontaneous revertant of ts110. An additional protein of Mr 55000 from uninfected cells was recognized by this serum. Reactivity of the serum toward the v-mos-containing proteins and the 55K protein was completely inhibited by prior incubation with free peptide. The 55K protein was not recognized by antisera made from synthetic peptides prepared from the C-terminal eight or 12 amino acids of v-mos.

Introduction of sequences encoding functional human adenosine deaminase into mouse cells using a retroviral shuttle system

A retroviral packaging system was used to generate a murine virus carrying sequences encoding human adenosine deaminase (ADA). To this end, human ADA cDNA was inserted into the retroviral shuttle vector pZIP-NeoSV(X)1. This vector provides all of the cis-acting sequences necessary for the efficient packaging and transmission of the viral genome as well as a selectable gene for G418 resistance. Transfection of this recombinant plasmid into cells that provide essential virus products (ψ-2 cells) yielded cell Unes that stably produced virions carrying the coding sequence of human ADA. We have used these virions to infect NIH3T3 cells, which after 48 h synthesized catalytically active human ADA. Furthermore, G418-resistant cell Unes were obtained from the virus-infected NIH3T3 ceUs that stably produced the human ADA enzyme.

Genetic organization and cloning of kirsten murine sarcoma virus DNA

Summary The Kirsten murine sarcoma virus genome is a recombinant molecule derived from the Kirsten murine leukemia virus genome and sequences transduced from a normal rat cell. We show here that leukemia virus derived sequences comprise the 5′ terminal 0.5 kilobasepairs (kbp) and the 3′ terminal 1.2 kbp of the linear 6.5 kbp sarcoma virus DNA. The parental leukemia virus thus contributes the long terminal repeat sequences (0.5 kbp) and some additional 3′ sequences to the sarcoma virus genome. Using DNA synthesised by detergent-disrupted virions a 4.6 kbp SmaI fragment containing the rat-specific sequences of the sarcoma virus was cloned into the plasmid pAT153. This clone was then used as a sarcoma virus specific probe to clone full sized circular viral DNA from infected NIH 3T3 cells.

Conversion of restrictive mouse cells to permissiveness during sequential and mixed double infection by murine leukemia viruses

Abstract Conversion from restrictive (two-hit) to permissive (one-hit) kinetics was observed when N- or B-tropic murine leukemia viruses were titrated on mouse embryo fibroblasts of nonpermissive Fv-1 genotype that had previously been nonproductively infected with the same virus at an average multiplicity of one. The effect was transient, disappearing within about 24 hr after the first infection, and was abrogated by exposure of the first virus preparation to increasing doses of ultraviolet light, with a D 37 inactivation dose of 2550 ergs/mm 2 , about three times that for inactivation of viral replication. Prior infection of nonpermissive mouse cells with the NZB xenotropic virus did not alter their restrictive response to later infection with ecotropic viruses. Low multiplicity infection of NIH 3T3 cells with a B-tropic virus, followed by treatment with iodoeoxyuridine, failed to induce productive infection by endogenous or exogenous virus. Cells of F 1 hybrid Fv-1 genotype, which are restrictive for both N- and B-tropic viruses, were converted to permissiveness for virus of either tropism after low multiplicity infection with virus of the opposite tropism. No evidence of NB-tropic recombinant progeny could be detected under these experimental conditions. The implications of these experimental observations with respect to the mechanism of restriction governed by the Fv-1 gene are discussed.