Following entry into the cytoplasm of the host cell, reverse transcription of the retroviral RNA genome into doublestranded viral DNA occurs within large nucleoprotein complexes derived from the core of the infecting virion, termed preintegration complexes (PICs). These complexes must then gain access to host cell chromosomes within the nucleus to achieve stable integration. Human immunodeficiency virus type 1 (HIV-1) can infect certain types of nondividing cells (5,22,46,74). Although the ability of lentiviruses to infect nondividing cells has been exploited in the development of vectors for human gene therapy, their application is limited by safety concerns. In contrast, nuclear accumulation of Moloney murine leukemia virus (Mo-MuLV) viral DNA increases only after infected cells pass through mitosis (61). This property limits the range of cells and target tissues that can use murinebased retroviral vectors for gene transfer or gene therapy.Many studies put forth the model that HIV-1 PICs are actively imported into the nucleus. In support of this model, nuclear localization signals (NLSs) have been reported on a number of viral proteins that comprise the HIV-1 PIC. The role or roles that these sequences play in viral replication, however, remain unclear. In addition, a cis-acting viral DNA structure generated during lentivirus-specific reverse transcription, termed the central DNA flap, may also be involved in the nuclear import of the HIV-1 genome (77). To date, no NLSs have been identified in MuLV PIC components. This suggests that it is the disassembly of the nuclear envelope during mitosis that renders the chromosomes accessible to the MuLV PIC.Interestingly, the avian sarcoma virus (ASV), which has a novel NLS sequence (44, 45), presents an intermediate phenotype in which mitosis-independent replication can occur, but at a lower efficiency than HIV-1 (31).Previous analysis indicated various molecular tags placed at the C terminus of the Mo-MuLV integrase (IN) protein could be incorporated into viral particles when expressed within the context of a replication-competent virus. IN proteins tagged with either six-His or hemagglutinin (HA) epitope tags were shown to be genetically stable during replication. In contrast, insertion of the SV40 large T-antigen NLS (SV40 NLS) was not stable, resulting in the selection and propagation of viruses in which the basic NLS sequence was disrupted (65). This study examines the stability of alternative NLS sequences within Mo-MuLV IN and the influence the NLS sequences have on the nuclear transport properties of Mo-MuLV. A similar approach to alter nuclear transport properties was reported to be successful for spleen necrosis virus (SNV), in which the efficiency of infecting nondividing cells was altered by incorporating an HIV-1 matrix (MA) protein NLS sequence into the SNV MA protein (56).Expression of IN and IN-SV40 NLS proteins. The effects of inserting the SV40 NLS into M-MuLV IN were first examined in an IN expression system in the absence of additional viral protein...

Foamy viruses are non-pathogenic retroviruses and represent a tool for vector development. For gene therapy applications and for analyses of viral protein composition infectious particles need to be purified, which has been difficult for foamy viruses in the past. Here, we describe a novel, simple, and fast purification method for prototype foamy viruses with high purity using size exclusion and affinity chromatography. More than 99,9% of the contaminating proteins were removed. The purified viruses were used to determine the amount of the incorporated Pol protein relative to Gag. The determined Gag to Pol PR-RT ratio of 30:1 confirmed previous studies suggesting FV virions encapsidate fewer number of Pol molecules than orthoretroviruses.