Infection of mice with exogenous ecotropic murine leukemia viruses (MuLVs) or expression of endogenous ecotropic MuLVs in certain mouse strains results in the generation of host-range variants derived by recombination with endogenous retroviral sequences. Recombination invariably involves the substitution of the receptor binding region of the exogenous virus with the env gene of endogenous polytropic retroviruses. These variants utilize a different cell surface receptor for infection and are capable of infecting cells from several species compared to ecotropic MuLVs that only infect murine cells; hence the designation, polytropic MuLVs. The induction of many diseases in mice by MuLVs involves the participation of polytropic MuLVs. These include the induction of proliferative, immunological and neurological disorders. Our earlier studies indicate that the interactions of ecotropic and polytropic MuLVs in the host play a major role in facilitating pathogenesis. This is apparent in mixed infections of ecotropic and polytropic MuLVs generated by recombination of exogenous retroviruses with endogenous retroviruses of the host, as well as in mixed infections generated by inoculation of mixtures of ecotropic and polytropic MuLVs. In the latter case inoculation of mice with retroviruses of different properties profoundly affects the replication of the viruses and the outcome of the infection. These included a highly significant delay in the induction of proliferative disease with one polytropic MuLV and a profound synergistic effect resulting in the abrupt development of a neurological disease with another polytropic isolate. In both instances the polytropic virus load in the co-inoculated mice was markedly enhanced while the level of the ecotropic MuLV was unchanged. Furthermore, the polytropic MuLV was nearly completely pseudotyped within ecotropic virions in co-inoculated mice. There are a number of possible mechanisms that could facilitate the profound in vivo amplification of the polytropic MuLVs. These may include enhanced spread of the virus due to pseudotyping within ecotropic virions or possibly transactivation of the polytropic virus in co-infected cells. To examine these questions in a less complex system we extended these studies to examine mixed retrovirus infections of an in vitro cell line. In vitro studies of ecotropic and polytropic mixed infections mirror many of the in vivo observations. The polytropic genome is highly pseudotyped within ecotropic virions and the replication of the polytropic virus is highly elevated compared to cells infected with the polytropic MuLV alone. Furthermore, the ecotropic infectivity remains unaltered. Recent studies indicate that the amplification of infectious polytropic MuLV in chronically infected cells is not uniform. We have found that the infected cultures are comprised of a heterogeneous mixture of cells, some of which release high levels of infectious viruses and some which release only trace or undetectable levels of infectious polytropic MuLV. Upon infection of the clones with an ecotropic virus, polytropic infectivity increased to high levels even with those clones that released only trace or undetectable levels of infectivity. In 2016 we have continued our investigations of individual clonal cell lines infected with the polytropic MuLV. Assessment of the specific infectivitys of the released virions revealed large differences that correlate with the level of the Env protein in the virions. A cell line that released only trace levels of infectivity released defective virions that were nearly devoid of the Env SU protein. The deficiency in SU is compensated by superinfection with an ecotropic MuLV which provides the Env proteins and increases the specific infectivity of Fr98-containing MuLVs upon superinfection. This deficiency is also circumvented by reinfection with a Fr98-RFP that encodes the entire Fr98 genome indicating that the deficiency in not an inability of the clonal cell line itself to properly assemble the polytropic Env protein. Our results suggest a unique context of the initial polytropic virus infection leading to a state that results in the suppression of Env protein levels in the cell and precludes the efficient incorporation the polytropic Env protein into progeny virions. This can result in a non-productive state characterized by the release of non-infectious virions containing retroviral RNA transcripts. This non-productive state is reversible by superinfection with a second retrovirus and thus represents a latent retroviral infection distinct from latency characterized by transcriptional silencing. We have observed decreases in the infectious virus production by some of our clonal lines during the course of these experiments. One of the clones after prolonged passage releases no detectable infectious particles, yet responds to superinfection by the ecotropic F-MuLV or reinfection by Fr98-RFP. This suggests a progressive and perhaps controlled mechanism of infectious virus silencing that may have important roles in in vivo retroviral infections as well as in the expression of endogenous retroviruses.
