A Protein Antagonist of Activation-Induced Cytidine Deaminase Encoded by a Complex Mouse Retrovirus

Abstract

Complex human-pathogenic retroviruses cause high morbidity and mortality worldwide, but resist antiviral drugs and vaccine development due to evasion of the immune response. A complex retrovirus, mouse mammary tumor virus (MMTV), requires replication in B and T lymphocytes for mammary gland transmission and is antagonized by the innate immune restriction factor murine Apobec3 (mA3). To determine whether the regulatory/accessory protein Rem affects innate responses to MMTV, a splice-donor mutant (MMTV-SD) lacking Rem expression was injected into BALB/c mice. Mammary tumors induced by MMTV-SD had a lower proviral load, lower incidence, and longer latency than mammary tumors induced by wild-type MMTV (MMTV-WT). MMTV-SD proviruses had many G-to-A mutations on the proviral plus strand, but also C-to-T transitions within WRC motifs. Similarly, a lymphomagenic MMTV variant lacking Rem expression showed decreased proviral loads and increased WRC motif mutations relative to those in wild-type-virus-induced tumors, consistent with activation-induced cytidine deaminase (AID) mutagenesis in lymphoid cells. These mutations are typical of the Apobec family member AID, a B-cell-specific mutagenic protein involved in antibody variable region hypermutation. In contrast, mutations in WRC motifs and proviral loads were similar in MMTV-WT and MMTV-SD proviruses from tumors in AID-insufficient mice. AID was not packaged in MMTV virions. Rem coexpression in transfection experiments led to AID proteasomal degradation. Our data suggest that rem specifies a human-pathogenic immunodeficiency virus type 1 (HIV-1) Vif-like protein that inhibits AID and antagonizes innate immunity during MMTV replication in lymphocytes. IMPORTANCE Complex retroviruses, such as human-pathogenic immunodeficiency virus type 1 (HIV-1), cause many human deaths. These retroviruses produce lifelong infections through viral proteins that interfere with host immunity. The complex retrovirus mouse mammary tumor virus (MMTV) allows for studies of host-pathogen interactions not possible in humans. A mutation preventing expression of the MMTV Rem protein in two different MMTV strains decreased proviral loads in tumors and increased viral genome mutations typical of an evolutionarily ancient enzyme, AID. Although the presence of AID generally improves antibody-based immunity, it may contribute to human cancer progression. We observed that coexpression of MMTV Rem and AID led to AID destruction. Our results suggest that Rem is the first known protein inhibitor of AID and that further experiments could lead to new disease treatments.

Unconventional ER to Endosomal Trafficking by a Retroviral Protein

“…Rem is a precursor protein that is directed for translation to the ER membrane where signal peptidase generates SP and the glycosylated Rem C-terminal product (Rem-CT) (Fig. 1A) (7,9,10). SP is then retrotranslocated from the ER membrane to the cytosol using the p97 ATPase (8,9).…”

“…Nuclear import of SP allows binding to the cis-acting Rem-responsive element (RmRE) found in all known MMTV mRNAs to facilitate nuclear export and post-export functions (2-4, 8, 9). Our recent studies show that loss of Rem C-terminal sequences antagonize the mutagenic effects of Apobec cytidine deaminases, particularly by proteasomal degradation of activation-induced cytidine deaminase (AID) during MMTV replication in lymphocytes (10). Since Apobec proteins are believed to function in both the cytoplasm and nucleus (101), determination of Rem-CT cellular localization was key to understanding its role in Apobec antagonism.…”

“…BFA treatment, which collapses the Golgi cisternae into the ERGIC, may lead to Rem-CT proteolysis. This model leaves many unanswered questions, but suggests that uncleaved Rem, rather than Rem-CT, may be the antagonist of Apobec family enzymes (10).…”

“…The median value of the colocalization coefficient for wild-type Rem-CT was ~0.5, whereas the mutant coefficient was significantly higher (~0.8). As previously reported, higher levels of transfected Rem-T7 expression plasmids led to abundant detection of both uncleaved Rem precursor as well as cleaved Rem-CT (10). Therefore, we performed Western blots of cell lysates from these transfections using T7-specific antibody.…”

“…Thus, SP has a function similar to human immunodeficiency virus type 1 (HIV-1)-encoded Rev (4). Our recent experiments indicate that Rem and/or Rem-CT act as accessory factors in vivo to counteract Apobec-mediated restriction of MMTV replication, particularly to antagonize the mutagenic activity of activation-induced cytidine deaminase (AID) (10). Since protein localization and function are tightly linked, the exact localization and trafficking of Rem-CT within host cells will contribute toward understanding its activity.…”