Interplay between MMTV and APOBEC3 proteins

Host organisms have adopted several strategies to block virus infections. Beside the innate and adaptive immunities the virus infection can be restricted due to the presence of intracellular inhibitory factors. The presence or absence of these "intracellular immunity" determines the permissiveness of the host to virus infection. Recently, a number of intrinsic intracellular anti-retroviral resistance mechanisms have been described. For example, post entry block of retrovirus infection has been shown to be mediated by the tripartite motif protein 5 alpha (TRIM5a). Another cellular factor, tetherin, inhibits release of retrovirus particles from the surface of infected cell. A growing number of retroviruses and endogenous mobile retroelements have been shown to be inactivated by members of the APOBEC3 (A3; apoliprotein B mRNA-editing enzyme catalytic polypeptide) family of cytidine deaminases. A3 proteins cause extensive mutations of nascent retroviral genomes by deamination of cytidine residues during reverse transcription. A Betaretrovirus, mouse mammary tumor virus (MMTV) that has been proposed to be involved in some human diseases such as human breast cancer, T-cell lymphoma and primary biliary cirrhosis, has also recently been shown to be inhibited by A3 proteins. Although MMTV has been the first retrovirus that has been demonstrated to be affected by human and mouse cytidine deaminases in vivo, the mechanism by which these proteins exert their function on the virus remains elusive. Based on our preliminary data and results of others, it seems, however, that MMTV infection is antagonized by a citidine-to-uracil editing independent means, recently described to inhibit, to some extent, also other retroviruses. During the course of the proposed project we would like to gain more insights into the A3-mediated mechanism counteracting MMTV infection. More specifically, we wish to assess the hypermutation levels, accumulation of reverse transcription products, nuclear imports of viral DNA, levels of provirus integrations and accumulations of aberrant autointegration products in the presence and absence of A3 polypeptides. MMTV is known as a simple retrovirus devoid of accessory proteins such as Vif of HIV-1 that protects HIV-1 from APOBEC3 proteins. Therefore, it is far from clear how simple retroviruses lacking a homologue of Vif avoid the deleterious effect of A3 proteins. In hopes of clarifying this question we plan to perform a site directed mutagenesis on selected regions within the MMTV genome (without affecting expression of structural genes) and analyze if the introduced mutations influence the replication potential of the virus in the presence of A3s. We believe that the proposed project would be beneficial not only to shed more light on the basic biology of MMTV but the findings obtained on the basis of this study may help to develop new strategies for therapy of retroviral infections (including HIV-1) as well as for treatment of devastating human diseases such as breast cancer and primary biliary cirrhosis.

Host organisms have adopted several strategies to block virus infections. Beside the innate and adaptive immunities the virus infection can be restricted due to the presence of intracellular inhibitory factors. The presence or absence of these intracellular immunity determines the permissiveness of the host to virus infection. One important family of restriction factors represents the apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide (APOBEC) family of proteins. The APOBEC3 in mouse and APOBEC3G in human cells catalyzes cytidine deamination of single stranded DNA or RNA substrates. In retrovirus-infected cells APOBEC proteins are encapsidated into viral particles budding from cells and cause extensive cytidine-to-uracil editing during reverse transcription. Viruses evolved counteraction mechanisms allowing virus replication in the APOBEC-expressing cells. These include exclusion of APOBEC proteins from virions and a virus-factor (Vif)-induced proteosomal degradation of APOBEC proteins in infected cells. We investigated the sensitivity of a prototypic betaretrovirus mouse mammary tumour virus (MMTV) to APOBEC3 proteins. Using a novel high titer MMTV-based vector system we found that MMTV is less sensitive to murine and human APOBEC3 proteins than HIV?Vif vectors. Trans-complementation experiments using various MMTV-based expression constructs and HIV?Vif as target vector showed that MMTV does not encode a functional analogue of the HIV-1 Vif directing proteosomal degradation of APOBEC3 proteins. Furthermore, the lower sensitivity to the APOBEC3 proteins correlated with lower level of cytidine-to-uracil mutation during reverse transcription that was observed despite of equivalent levels of APOBEC3 proteins being packaged into HIV?Vif and MMTV virions. An F120L mutant carrying Phe-to-Leu substitution at the DNA polymerase domain of reverse transcriptase (Phe 120 is homologous to Phe156 of the HIV-1 RT in the dNTP binding domain) exhibited an increased sensitivity to APOBEC3 proteins. Taken together, our results indicate that relative insensitivity to APOBEC3 proteins is not mediated by exclusion of APOBEC3 from virions or by a virus factor-mediating degradation of APOBEC3 proteins. Instead, our data suggest that the lower extent of MMTV inhibition in the presence of APOBEC3 is an intrinsic property of the MMTV reverse transcriptase that does not allow efficient APOBEC3-mediated attack of the nascent negative sense single stranded viral DNA during reverse transcription.