Approximately 8% of the genomes of mammals, including humans and mice, are comprised of retroviral elements acquired by infection of germ line cells during the course of evolution. Retroviral insertions in our genome number about 40,000. Most endogenous retrovirus elements are defective for replication however several contain one or more viral genes that are expressed during development and during certain physiological or pathological conditions. Little is known about the control of retrovirus expression or the influence of such expression on the physiology or pathology of the host. Polytropic MuLVs are an extensively investigated group of endogenous retroviruses that give rise to recombinant murine leukemia viruses (MuLVs) in mice. Upon infection of mice with exogenous ecotropic MuLVs, members of this group undergo recombination to generate new MuLVs with an altered infectious host range. Recombination requires transcription of the endogenous retroviruses. Although the endogenous polytropic proviruses are transcribed; replication of the endogenous polytropic viruses in the absence of recombination has not been observed. This may, in many cases, reflect defects such as point mutations or deletions in the endogenous viral genome but may also be influenced by the activity of various restriction factors. The fact that exogenous MuLVs are capable of replicating in mice indicates that they have evolved mechanisms to circumvent the activity of at least some of the restriction factors such as the murine APOBEC3 (mA3). Thus, exogenous retroviruses might facilitate,through complementation, active replication of endogenous retroviruses. We have found that infection of mice by an exogenous virus results in the infectious transfer of complete endogenous proviral genetic sequences. This includes proviruses which are severely defective and possess large deletions as well as proviruses that are full-length. Furthermore, the transferred sequences are transcribed and packaged into virions released from the newly infected cells. At early times after infection with the Friend MuLV, packaging and transfer of intact endogenous retroviruses is much more prevalent than recombination. Endogenous retroviruses are transferred as early as one day after infection. Thus, the transcripts are captured within a single in vivo replication cycle and originate from among the initially infected host cells. The level of complete endogenous proviruses that are mobilized during the course of disease is close to the level of recombinanat polytropic viruses that have been implicated in disease. The mobilization of intact endogenous retroviruses is unprecedented and may have important implications for the involvement of endogenous retroviruses in disease processes. In 2015 we have extended our observations to further characterize the endogenous viruses mobilized after infection by exogenous retroviruses. We have found that the mobilized endogenous viruses appear to be specifically limited to endogenous polytropic proviruses at the exclusion of other endogenous retroviral elements. Furthermore, structures of some of the mobilized endogenous viruses exhibit frequent recombination among them indicating their active replication, likely as a result of infection of the host with an exogenous retrovirus. The endogenous polytropic proviruses are comprised of two structural subclasses termed Polytropic (PT) and Modified Polytropic (mPT). We observed a distinct shift in the subclass of proviruses from the mPT subclass to the PT subclass of polytropic proviruses detected during the course of infection. These results suggest the spread of infection to cells expressing different classes of polytropic viruses or an alteration in the expression of the endogenous proviruses in infected cells. Exogenous mouse retroviruses encode a glycosylated gag protein (gGag) originating from an alternate translation start site upstream of the methionine start site of the gag structural polyproteins. The functions of gGag remain unclear, but mutations that eliminate its synthesis severely impede in vivo replication of the virus with little effect on replication in fibroblastic cell lines. APOBEC3 proteins have evolved as innate defenses against retroviral infections. Both mice and humans express APOBEC3 proteins that have cytidine deaminase activity leading to hypermutation of viral transcripts and inactivation of infecting retroviruses. HIV encodes the VIF protein to evade human APOBEC3G (hA3G), however mouse retroviruses do not encode a VIF homologue and it has not been understood how they evade mouse APOBEC3 (mA3). We have found that a mouse retrovirus utilizes its glycosylated gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice implicating a novel role of gGag in circumventing the action of APOBEC3 in vivo. In 2015 we have continued to focus on the elucidation of the mechanism by which the gGag protein abrogates the action of APOBEC3. We have found that mA3 packaged at very high levels in wild-type virions results in a loss of specific infectivity, a loss in the specific enzymatic activity of the viral polymerase accompanied by a substantial increase in the overall mutation rate. The mutagenic activity of mA3 in wild-type as well as gGag-deficient viruses does not include G to A hypermutation and is unlikely to involve cytidine deamination. We have found that the ecotropic MuLV, AKV, undergos G to A hypermutation with a target sequence indicative of the action of mA3. However, hypermutation of AKV does not require the augmentation of mA3 in cells. Furthermore, hypermutation occurs in only a portion of the virus transcripts suggesting that a restriction factor is present in only a portion of the virions. These results suggest that AKV is exquisitely susceptible to mA3 or another restriction factor expressed at trace levels in the cells.
Showing the most recent 10 out of 12 publications
