Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Integration of murine leukemia virus DNA depends on mitosis.

In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integration machinery. When infected cells traverse mitosis, there is a sharp increase in nuclear accumulation of viral DNA. The dependence of integration on mitosis may therefore be due to a requirement for mitosis and nuclear envelope breakdown for entry of the viral integration complex into the nucleus.     

The HIV-1 passage from cytoplasm to nucleus: the process involving a complex exchange between the components of HIV-1 and cellular machinery to access nucleus and successful integration

The human immunodeficiency virus 1 (HIV-1) synthesizes its genomic DNA in cytoplasm as soon as it enters the cell. The newly synthesized DNA remains associated with viral/cellular proteins as a high molecular weight pre-integration complex (PIC), which precludes passive diffusion across intact nuclear membrane. However, HIV-1 successfully overcomes nuclear membrane barrier by actively delivering its DNA into nucleus with the help of host nuclear import machinery. Such ability allows HIV-1 to productively infect non-dividing cells as well as dividing cells at interphase. Further, HIV-1 nuclear import is also found important for the proper integration of viral DNA. Thus, nuclear import plays a crucial role in establishment of infection and disease progression. While several viral components, including matrix, viral protein R, integrase, capsid, and central DNA flap are implicated in HIV-1 nuclear import, their molecular mechanism remains poorly understood. In this review, we will elaborate the role of individual viral factors and some of current insights on their molecular mechanism(s) associated with HIV-1 nuclear import. In addition, we will discuss the importance of nuclear import for subsequent step of viral DNA integration. Hereby we aim to further our understanding on molecular mechanism of HIV-1 nuclear import and its potential usefulness for anti-HIV-1 strategies.     

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.     

An Avian Sarcoma/Leukosis Virus-Based Gene Trap Vector for Mammalian Cells

RCASBP-M2C is a retroviral vector derived from an avian sarcoma/leukosis virus which has been modified so that it uses the envelope gene from an amphotropic murine leukemia virus (E. V. Barsov and S. H. Hughes, J. Virol. 70:3922-3929, 1996). The vector replicates efficiently in avian cells and infects, but does not replicate in, mammalian cells. This makes the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems. Here we describe the development of a derivative of RCASBP-M2C, pGT-GFP, that can be used in gene trap experiments in mammalian cells. The gene trap vector pGT-GFP contains a green fluorescent protein (GFP) reporter gene. Appropriate insertion of the vector into genes causes GFP expression; this facilitates the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of GFP. With this vector, we have generated about 90 gene-trapped lines using D17 and NIH 3T3 cells. Five trapped NIH 3T3 lines were selected based on the distribution of GFP in cells. The cellular genes disrupted by viral integration have been identified in four of these lines by using a 5' rapid amplification of cDNA ends protocol.     

Analysis of cellular integration sites in avian sarcoma virus infected duck embryo cells.

The cellular sites of integration of avian sarcoma virus (ASV) have been examined in clones of duck embryo cells infected with the Bratislava 77 strain of ASV using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled ASV complementary DNA probes. DNA prepared from 11 clones of duck embryo cells infected with the Bratislava 77 strain of ASV was digested with the restriction enzymes HpaI, which cleaves once within the viral genome, and Hind III, which cleaves twice within the viral genome, and the virus-cell DNA juncture fragments were resolved by agarose gel electrophoresis. Analysis of the virus-cell junctures present in individual ASV-infected duck embryo clones revealed that all clones contain at least one copy of nondefective proviral DNA with some clones containing as many as 5 to 6 copies of proviral DNA. A comparison of the virus-cell juncture fragments present in different ASV-infected clones showed that each clone contains a unique set of virus-cell junctures. These data suggest that ASV DNA can integrate at multiple sites within the duck embryo cell genome and that these sites appear to be different as defined by digestion with the restriction enzymes HpaI and HindIII.     

Nuclear import of moloney murine leukemia virus DNA mediated by adenovirus preterminal protein is not sufficient for efficient retroviral transduction in nondividing cells

Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G(1)/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration.     

Genetic evidence that the avian retrovirus DNA endonuclease domain of pol is necessary for viral integration.

We used in vitro mutagenesis in the 3' region of the avian retrovirus polymerase (pol) gene to genetically define the role of the DNA endonuclease domain. In-frame insertional mutations, which were dispersed throughout the 5' region of pp32, produced a series of five replication-deficient mutants. In contrast, a single point mutant (Ala----Pro) located 48 amino acids from the NH2 terminus of pp32 exhibited a delayed replication phenotype. Molecular analysis of this mutant demonstrated that upon infection it was capable of synthesizing both linear and circular species of unintegrated viral DNA. The levels of unintegrated viral DNA present in cells infected with the mutant virus were several times greater than wild-type levels. Quantitation of the amount of integrated viral genomes demonstrated that the mutant virus integrated viral DNA one-fifth as efficiently as wild-type virus. This single point mutation in the NH2 terminus of pp32 prevented efficient integration of viral DNA, with no apparent effect on viral DNA synthesis per se. Thus, the DNA endonuclease domain has been genetically defined as necessary for avian retrovirus integration.     

HMG protein family members stimulate human immunodeficiency virus type 1 and avian sarcoma virus concerted DNA integration in vitro

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571-3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.     

Lentivirus gene transfer in murine hematopoietic progenitor cells is compromised by a delay in proviral integration and results in transduction mosaicism and heterogeneous gene expression in progeny cells

Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin(-) c-kit(+) Sca1(+) primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% +/- 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42. 0% +/- 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 +/- 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 +/- 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP(+) lentivirus vector-transduced colonies revealed vector PCR(+) GFP(+) (42%), vector PCR(-) GFP(-) (46%), and vector PCR(+) GFP(-) (13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior to vector integration.     

Suppression of multiplication of avian sarcoma virus by rapid spread of transformation-defective virus of the same subgroup.

We have tested the hypothesis that some transformation-defective (td) viruses grow faster than the avian sarcoma viruses (ASV) from which they are derived, resulting in establishment of interference by the td virus and suppression of the ASV multiplication. Using an ASV of subgroup A (ASV-A) that does not contain td virus and an independently isolated tdASV-A, we performed separate and mixed infections to test this hypothesis. At multiplicities of 1 or less, tdASV alone grew to higher titers and more rapidly than ASV alone. In mixed infections at low multiplicities that allowed spread of progeny virus, when as little as 10% of the virus inoculum was td virus, there was an excess of td virus by 2 days after infection and a decrease in the titer of ASV relative to a control infection with no td virus. In mixed infections at high multiplicities which minimized spread of progeny virus, there was no excess of td virus and the titer of ASV was not decreased relative to the control infection with no td virus. These data support the hypothesis that we proposed and indicate that deletions in the ASV src gene may not be a high-frequency event. We also present data concerning the amounts of unintegrated viral DNA found after the separare and mixed infections. There was no simple correlation between the amounts of unintegrated viral DNA early after infection and the titers of virus produced, indicating perhaps that virus production was determined by integrated viral DNA.