Transduction of Interphase Cells by Avian Sarcoma Virus

Abstract

It has been generally believed that oncoretroviruses are dependent on mitosis for efficient nuclear entry of viral DNA. We previously identified a nuclear localization signal in the integrase protein of an oncoretrovirus, avian sarcoma virus (ASV), suggesting an active import mechanism for the integrase-DNA complex (G. Kukolj, R. A. Katz, and A. M. Skalka, Gene 223:157-163, 1998). Here, we have evaluated the requirement for mitosis in nuclear import and integration of ASV DNA. Using a modified ASV encoding a murine leukemia virus amphotropic env gene and a green fluorescent protein (GFP) reporter gene, DNA nuclear import was measured in cell cycle-arrested avian (DF-1) as well as human (HeLa) and mouse cells. The results showed efficient accumulation of nuclear forms of ASV DNA in ␥-irradiation-arrested cells. Efficient transduction of a GFP reporter gene was also observed after infection of cells that were arrested with ␥-irradiation, mitomycin C, nocodazole, or aphidicolin, confirming that nuclear import and integration of ASV DNA can occur in the absence of mitosis. By monitoring GFP expression in individual cells, we also obtained evidence for nuclear import of viral DNA during interphase in cycling cells. Lastly, we observed that ASV can transduce postmitotic mouse neurons. These results support an active nuclear import mechanism for the oncoretrovirus ASV and suggest that this mechanism can operate in both nondividing and dividing cells.Early events in the retroviral replication cycle include reverse transcription of the viral RNA and integration of the resulting viral DNA genome into the host cell chromosomes (6, 12). These two steps are catalyzed by the viral enzymes reverse transcriptase (RT) and integrase (IN), which are carried into the cell within the virion capsid. In addition to the enzymatic steps, these early events require trafficking of the viral DNA, which is synthesized in the cytoplasm, to sites of integration in the host cell chromosomes in the nucleus. The precursor to the integrated viral DNA is a linear doublestranded DNA which is found in a subviral preintegration complex that contains IN, as well as other host and viral proteins. It is likely that all retroviruses utilize active mechanisms (as opposed to passive diffusion) for subcellular trafficking of viral DNA, and such movement may be facilitated by interactions between the preintegration complex and host cell factors or structures. As efficient and stable expression of viral DNA requires integration into the host chromosome, the transport of viral DNA to an integration site is essential to complete the early steps in infection. Integration of the viral DNA marks a transition to late steps that include synthesis of viral RNAs and proteins, followed by assembly and budding of progeny particles.The nuclear membrane is a potential barrier for movement of retroviral DNA (in the preintegration complex) to the chromosomal integration sites. In dividing cells, which are progressing through the cell cycle (G1 3 S 3 G 2 3 M), the nuclear me...

Effects of cell cycle status on early events in retroviral replication

“…The discovery that HIV-1 could complete early events in some non-cycling cells and that HIV-1 DNA could enter the nucleus through the NPC [Weinberg et al, 1991;Bukrinsky et al, 1992], appeared to distinguish HIV-1 from the prototype ''oncoretrovirus,'' MLV, which required mitosis. However, recent studies with ASV have demonstrated its capacity for mitosis-independent integration [Hatziioannou and Goff, 2001;Katz et al, 2002Katz et al, , 2003Greger et al, 2004], consistent with the earlier findings [Humphries et al, 1981]. Thus, mitosis-independent integration of viral DNA in non-cycling cells is not limited to HIV and lentiviruses.…”

“…There has been considerable focus on HIV NPCmediated import. However, as noted above, ASV can also infect some non-cycling cells [Hatziioannou and Goff, 2001;Katz et al, 2002;Greger et al, 2004] and may therefore also encode multiple signals that promote nuclear entry through the NPC.…”

“…These seemingly unique properties of lentiviruses have been thought to be attributable, at least in part, to the additional or novel gene products. Although these widely held notions have recently been shown to be inappropriately broad [Hatziioannou and Goff, 2001;Katz et al, 2002], it is clear that lentivirus-based vectors show significantly higher potential for gene delivery to non-cycling cells than the traditional MLV-based (oncoretroviral) vectors [Naldini et al, 1996]. However, HIV-based vector systems that have been stripped of all auxiliary and regulatory and proteins can still complete early events in non-cycling cells [Trono, 2000].…”

“…It was later demonstrated that low nucleotide pools are also limiting for reverse transcription of HIV-1 [Gao et al, 1993]. However S-phase itself is not absolutely required for completion of early steps by a variety of retroviruses, indicating that all of the early steps in the retroviral life cycles can be completed successfully in non-cycling cells that are not expected to contain peak concentrations of dNTPs [Weinberg et al, 1991;Hatziioannou and Goff, 2001;Katz et al, 2002;Greger et al, 2004]. Furthermore, dNTP levels are relatively low in post-mitotic cells that have withdrawn from the cell cycle (G 0 ), such as macrophages [Terai and Carson, 1991], yet HIV can propagate in these cells.…”

Early Detection of a Two-Long-Terminal-Repeat Junction Molecule in the Cytoplasm of Recombinant Murine Leukemia Virus-Infected Cells

“…Indeed, infecting G 1 /S-arrested cells with MLV resulted in the detection of two-LTR circle junctions 8 h after the cells were released and underwent a complete cycle (28). Recently, a comparison between retrovirus vectors demonstrated that aphidicolin-arrested cells could be infected by avian sarcoma virus but not by MLV vectors (20,25). In these studies, circular viral DNA molecules were looked for 16 to 24 h post viral infection as an indicator of PIC nuclear translocation and productive infection.…”