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Previous reports have shown that retrovirus infection is inhibited in nonreplicating (stationary-phase [hereafter called stationary]) cells. Infection of stationary cells was shown to occur when the cells were allowed to replicate at times up to a week after infection, suggesting that an unintegrated retrovirus could persist in a form that was competent to integrate after release of the block to replication. However, those studies were complicated by the use of replication-competent virus, which can spread in the infected cells. We have used a replication-defective retrovirus vector to compare the efficiency of gene transfer in stationary and replicating rat embryo fibroblasts. In agreement with previous results, gene transfer was inhibited 100-fold in stationary versus replicating cells. In contrast to previously reported results, the block to infection could not be relieved by stimulating stationary cells to divide at times from 6 h to 10 days after infection. Thus, for successful retroviral infection, the infected cells must be replicating at the time of infection. These results have important implications for the use of retroviral vectors for gene transfer.
Mol Cell Biol 1990 Aug
PMID:Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. 237 Aug 65

Infection of Vero (monkey) cells by Germiston bunyavirus is highly cytopathic with cell lysis and virus production at a high titre, whereas infection of Aedes albopictus C6/36 (mosquito) cells leads, after an acute primary phase, to a persistent non-cytopathic infection with a loss in virus production. In this report we demonstrate that single-stranded RNA probes can be successfully used in an in situ hybridization assay to quantify viral expression during this persistent infection. The steady-state levels of viral S-RNA segment (genomic and messenger sense) during the acute phase were similar to those observed in lytically infected Vero cells, but appeared delayed. Both senses of S-RNA were detected throughout persistent infection but in lower amounts, in less than 10% of the cells and always in the cytoplasm of infected cells. The number of copies per cell of messenger sense S-RNAs remained low during persistent infection whereas a higher fluctuation was observed for genomic S-RNAs. In situ hybridization with specific stranded RNA probes provides both qualitative and quantitative informations, that can lead to a better understanding of virus-cell interactions.
Mol Cell Probes 1990 Aug
PMID:Quantitative in situ hybridization using strand specific RNA probes: expression of the bunyavirus Germiston S segment in mosquito cells. 240 48

BALB-/MK-2 mouse epidermal keratinocytes required epidermal growth factor for proliferation and terminally differentiated in response to high Ca2+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal growth factor dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal keratinocyte terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies.
Mol Cell Biol 1985 Dec
PMID:Members of the src and ras oncogene families supplant the epidermal growth factor requirement of BALB/MK-2 keratinocytes and induce distinct alterations in their terminal differentiation program. 242 28

Infection of A431 cells with vaccinia virus, or exposure to a mitogenic polypeptide secreted by vaccinia virus-infected cells, induces tyrosine phosphorylation of epidermal growth factor receptors.
Mol Cell Biol 1986 Jan
PMID:Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors. 243 Dec 67

We have constructed an IPTG-inducible plasmid which overexpresses oop RNA sequences in Escherichia coli. Infection of these transformed E. coli cells (SB221/pOOP5) with lambda+ phage produced clear plaques, whereas lambda+ infection of cells transformed with the plasmid vector (SB221/pJDC406) or the plasmid expressing the oop RNA transcript in the other orientation (SB221/pOOP9) gave rise to turbid plaques characteristic of lambda+. Calculations of the percentage of infected cells forming lysogens show a 6-fold decrease in the absence of isopropyl beta-D-thiogalactoside (IPTG) and a 20-fold decrease in the presence of IPTG for SB221/pOOP5 as compared to both SB221/pJDC406 and SB221/pOOP9. We have thus shown that the overexpression of oop RNA favors the lytic mode of lambda development.
Mol Gen Genet 1987 Nov
PMID:Overproduction of an antisense RNA containing the oop RNA sequence of bacteriophage lambda induces clear plaque formation. 244 89

Infection with the bacteriophage mutant Mu c+ gemts2 at 42 degrees C induces synchrony in cell division in cultures of Escherichia coli K12. This synchrony may last for several cycles and is not only due to selection since synchronization is observed even when bacterial survival to the infection is over 80% as in lysogens for Mu c+ gemts2. The mechanism by which synchrony is induced is not known, but since the product of Mu gene gem (previously called lig) has been shown to interact with the enzymatic system in the bacteria controlling the degree of DNA supercoiling, the phenomenon could be a consequence of this interaction.
Mol Gen Genet 1989 Jul
PMID:Synchronous division induced in Escherichia coli K12 by gemts mutants of phage Mu. 252 78

Continuous cultivation of peripheral blood lymphocytes from healthy sheep was carried out in vitro with the help of human recombinant interleukin-2. Lymphocytes were concurrently cultivated with the lethally X-rayed BLV-producing FLK culture cells. Electron microscopy and dot-blot hybridization established that sheep peripheral blood lymphocytes were infected with BLV and a full cycle of replication takes place in them. Infection of sheep leukocytes in vitro can be used to study the mechanisms of leukogenesis in vitro.
Mol Gen Mikrobiol Virusol 1989 Jan
PMID:[Infection of peripheral blood lymphocytes in sheep with bovine leukemia virus in vitro]. 254 21

Murine long-term bone marrow cultures that support B-lymphoid-cell development were infected with a helper-free retrovirus containing the v-fms oncogene. Infection of B-lymphoid cultures resulted in the rapid clonal outgrowth of early pre-B cells, which grew to high cell densities on stromal cell feeder layers, expressed v-fms-coded glycoproteins, and underwent immunoglobulin heavy-chain gene rearrangements. Late-passage cultures gave rise to factor-independent variants that proliferated in the absence of feeder layers, developed resistance to hydrocortisone, and became tumorigenic in syngeneic mice. The v-fms oncogene therefore recapitulates known effects of the v-abl and bcr-abl oncogenes on B-lineage cells. The ability of v-fms to induce transformation of early pre-B cells in vitro underscores the capacity of oncogenic mutants of the colony-stimulating factor-1 receptor to function outside the mononuclear phagocyte lineage.
Mol Cell Biol 1989 Sep
PMID:Early pre-B-cell transformation induced by the v-fms oncogene in long-term mouse bone marrow cultures. 255 Aug 8

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.
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PMID:Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector. 255 79

The bovine papillomavirus (BPV) type 1 E5 gene encodes a 44-amino-acid protein that can stably transform cultured rodent cells when expressed in the absence of all other viral genes. We have previously constructed a BPV-simian virus 40 recombinant virus (Pava-1) which efficiently expresses the BPV type 1 E5 gene in infected cells (J. Settleman and D. DiMaio, Proc. Natl. Acad. Sci. USA 85:9007-9011, 1988). Within 48 h of Pava-1 infection, the vast majority of mouse C127 cells underwent a dramatic morphologic transformation which was accompanied by cell proliferation. Infection of C127 cells made quiescent by contact inhibition and serum starvation caused a great induction of cellular DNA synthesis. These morphologic and mitogenic responses were proportional to the virus multiplicity of infection. Mutational analysis indicated that the E5 gene is both necessary and sufficient for these activities. Analysis of a variety of E5 missense mutants revealed a strong correlation between their phenotypes in the acute transformation assays following infection and in the stable focus-forming assay following transfection. Most of the defective mutants expressed normal levels of E5 protein following infection, indicating that their defective phenotypes are not due to the synthesis of an unstable protein. The failure to genetically resolve these E5 activities suggests that the ability of the E5 protein to cause acute morphologic transformation and reentry into the cell cycle may be intimately related to its ability to cause stable cell transformation and that these functions are probably mediated by a single biochemical activity of the E5 protein.
Mol Cell Biol 1989 Dec
PMID:Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated by a single activity of the bovine papillomavirus E5 protein. 255 1


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