Identification of envelope determinants of feline leukemia virus subgroup B that permit infection and gene transfer to cells expressing human Pit1 or Pit2.

The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116--8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.

Cell-type-specific regulation of the two foamy virus promoters.

The foamy virus (FV) genome contains two promoters, the canonical long terminal repeat (LTR) promoter, containing three consensus AP-1 binding sites, and an internal promoter (IP) within the env gene. We investigated the regulation of the two promoters in lytic and persistent infections and found that in the presence of a constitutive source of the viral transactivator protein Tas, transactivation of the LTR promoter and that of the IP differ. In lytic infections, both the LTR promoter and the IP are efficiently transactivated by Tas, while in persistent infections, the IP is efficiently transactivated by Tas, but the LTR promoter is not. Analysis of proteins expressed from the LTR promoter and the IP during infection indicated that IP transcription is more robust than that of the LTR promoter in persistently infected cells, while the opposite is true for lytically infected cells. Coculture experiments also showed that LTR promoter transcription is greatest in cells which support lytic replication. Replacement of much of the LTR promoter with the IP leads to increased viral replication in persistent but not lytic infections. We also found that the induction of persistently infected cells with phorbol 12-myristate 13-acetate (PMA) greatly enhanced viral replication and transcription from the SFVcpz(hu) (new name for human FV) LTR promoter. However, mutation of three consensus AP-1 binding sites in the FV LTR promoter did not affect viral replication in lytically or persistently infected cells, nor did the same mutations affect LTR promoter transactivation by Tas in PMA-treated cells. Our data indicate that differential regulation of transcription is important in the outcome of FV infection but is unlikely to depend on AP-1.

Historical perspective of foamy virus epidemiology and infection.

Foamy viruses (FV) are complex retroviruses which are widespread in many species. Despite being discovered over 40 years ago, FV are among the least well characterized retroviruses. The replication of these viruses is different in many interesting respects from that of all other retroviruses. Infection of natural hosts by FV leads to a lifelong persistent infection, without any evidence of pathology. A large number of studies have looked at the prevalence of primate foamy viruses in the human population. Many of these studies have suggested that FV infections are prevalent in some human populations and are associated with specific diseases. More recent data, using more rigorous criteria for the presence of viruses, have not confirmed these studies. Thus, while FV are ubiquitous in all nonhuman primates, they are only acquired as rare zoonotic infections in humans. In this communication, we briefly discuss the current status of FV research and review the history of FV epidemiology, as well as the lack of pathogenicity in natural, experimental, and zoonotic infections.

Multiple integrations of human foamy virus in persistently infected human erythroleukemia cells.

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete tas gene, which encodes the viral transactivator, and are not derived from Deltatas cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.