Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes mammary tumors in mice. MMTV is the only known complex murine retrovirus and encodes Rem, an HIV-1 Rev-like protein. Rem is a 301-amino-acid (33 kDa) protein that is cotranslationally targeted to the ER, where the first 98 amino acids constitute the signal peptide (SP). The SP is cleaved and retrotranslocated to the cytoplasm prior to nuclear entry. In this thesis, the results show that the presence of a leucine at position 71 allows more efficient cleavage of SP and increases Rem activity. Further, in Rem-transfected cells, the majority of SP appears in the nuclear fraction, consistent with fluorescent microscopy data. The C-terminal fragment of Rem (RemCT) is glycosylated in the ER and, although glycosylation sites are present outside the SP, mutations of both these sites abolish SP activity in a reporter assay. Indirect evidence suggests that unglycosylated RemCT is degraded by the proteasome, whereas glycosylated RemCT is likely secreted out of the cell. A variant of MMTV (TBLV) that lacks functional Sag and RemCT has been prepared and will be studied in mice to elucidate the role of RemCT in vivo. Development of an antibody to RemCT will allow tracking of the protein in virus-producing cells. Although there are many other similarities between complex retroviruses like HIV-1 and MMTV, current evidence suggests that Rem lacks an HIV Tat-like transactivator function. / text

Mouse mammary tumor virus (MMTV), a member of the betaretrovirus family, primarily induces mammary carcinomas in mice. Like human immunodeficiency virus (HIV), MMTV is a complex retrovirus that encodes a viral regulatory protein, Rem. Rem is a 33 kDa glycosylated protein containing an unusually long ER signal peptide (SP). MMTV SP contains all of the functional motifs for the nuclear export of MMTV unspliced/genomic RNA. SP activity requires binding to MMTV RNA. To characterize the minimal Rem-responsive element (RmRE) that overlaps the 3’ LTR, several deletion mutations were introduced in the MMTV-based reporter plasmid, pHMRluc. Results from these mutants in transient transfections revealed a 476-nt RmRE at the junction of the envelope gene and the 3’ LTR. RmRE function was not cell-type specific. The RmRE is predicted to have a complex secondary structure, similar to the Rev-responsive element (RRE) of HIV. Unlike the HIV RRE, the 3’ LTR RmRE occurs in all MMTV mRNAs, and Rem does not increase the export of unspliced RNA of the pHMRluc reporter vector. These results suggest that another RmRE near the 5’-end participates in export of MMTV genomic RNA, whereas the RmRE overlapping the 3’ LTR supports different Rem functions, such as translational regulation. Recent research has shown that SP directs Rem translation to the ER where Rem is cleaved and released into the cytoplasm. Rem mutants with ER signal peptidase cleavage site mutations completely lost function, and mutant proteins were highly unstable and mislocalized. Dominant-negative AAA ATPase p97 and Derlin-1 proteins, which are involved in the ER-associated degradation (ERAD) pathway, inhibited Rem function. Therefore, Rem is a precursor protein that is processed by ER signal peptidases. Rem then manipulates the ERAD system to retrotranslocate SP to the cytoplasm prior to nuclear entry and MMTV RNA binding. Unexpectedly, a commercial control shRNA expression vector, LK0.1, induced additional Rem, HIV-1 Rev and human T-cell leukemia virus type 1 Rex activity (called super-induction). Also, the LK0.1 vector increased protein expression levels of co-transfected genes, and the target of the shRNA was not critical. When the hairpin segment was deleted from LK0.1, the super-induction of Rem activity was greatly reduced. Deletion of cis-acting lentiviral segments also decreased protein expression levels. Although LK0.1 did not affect the levels of interferon-induced genes or eIF-2α phosphorylation, LK0.1 reduced the number of stress granules significantly. Therefore, LK0.1 may induce several cellular signaling pathways, leading to Rem super-induction. This study characterizes the minimal RmRE overlapping the 3’ MMTV LTR and reveals the unique processing of Rem and SP trafficking prior to nucleolar localization. Additional functions of MMTV Rem and other retroviruses may be discovered using studies of cellular events induced by LK0.1. / text

Die ER-assoziierte Proteindegradation (ERAD) ist Teil des Qualitätskontrollsystems am ER, um der Akkumulation von fehlgefalteten Proteinen im ER entgegenzuwirken. Hierbei werden ERAD-Substrate mit Hilfe von E3-Ligasen wie z.B. HRD1 ubiquityliert und anschließend durch den p97-Ufd1-Npl4 Komplex aus der ER-Membran extrahiert. Im Zytosol werden diese extrahierten Proteine vom 26S Proteasom abgebaut. Für die Retrotranslokation von ERAD- Substraten werden zudem die Membranproteine Derlin-1 und VIMP benötigt, welche mit p97 assoziieren und einen Proteinkomplex bilden. HERP ist ein ER-lokalisiertes Protein, dessen Synthese durch den UPR (unfolded protein response) als Antwort auf die Akkumulation von fehlgefalteten Proteinen im ER induziert wird. Dies deutet auf eine Rolle von HERP im ERAD hin. Interessanterweise besitzt HERP eine sogenannte UBL-Domäne. Für andere Proteine mit UBL-Domäne konnte eine Interaktion dieser Domäne mit dem Proteasom nachgewiesen werden. Daher kann angenommen werden, dass HERP ebenfalls mit dem Proteasom interagiert und dies zur ER- Membran rekrutiert, wo es für den Abbau von ERAD-Substraten benötigt wird. Das Ziel der vorliegenden Arbeit war es, die Rolle von HERP innerhalb des UPR zu ermitteln. Die hier präsentierten Daten zeigen, dass HERP essentiell für den Abbau des ERAD-Modell- Substrates CD3-delta ist. Somit hat HERP tatsächlich eine Rolle im ERAD. Außerdem wird eine direkte Interaktion von HERP mit der E3-Ligase HRD1 nachgewiesen. Es wird zudem gezeigt, dass HERP und HRD1 einen Proteinkomplex mit p97, Derlin-1 und eventuell auch mit VIMP bilden. Dieser ERAD Komplex ist folglich sowohl für die Ubiquitylierung als auch die Retrotranslokation von ERAD-Substraten verantwortlich und garantiert somit die effiziente Prozessierung von Proteinen aus dem ER. Zudem wird gezeigt, dass die UBL-Domäne von HERP im Gegensatz zu anderen UBL- Domänen nicht mit dem Proteasom interagiert. Somit kann nicht mehr davon ausgegangen werden, dass Proteasombindung eine Gemeinsamkeit aller Proteine mit UBL-Domäne ist. Dagegen wird eine Interaktion der UBL-Domäne von HERP mit dem deubiquitylierenden Enzym USP7 nachgewiesen. Dies deutet darauf hin, dass auch Deubiquitylierung eine wichtige Rolle im ERAD-Prozess spielt. / ER-associated protein degradation (ERAD) is part of the ER quality control system dealing with the accumulation of misfolded proteins in the ER. This process requires polyubiquitylation of ERAD substrates involving E3 ligases, such as HRD1, and their subsequent extraction from the ER membrane by the p97-Ufd1-Npl4 complex. Retrotranslocation of substrates into the cytosol for degradation by the 26S proteasome also involves the membrane proteins Derlin-1 and VIMP, which are associated with p97 to form a protein complex. The ER-resident protein HERP was shown to be upregulated by the unfolded protein response pathway (UPR) upon the accumulation of misfolded proteins in the ER. It was therefore considered to function in ERAD. Interestingly, HERP contains a UBL domain. In other proteins this domain facilitates an interaction with the proteasome, suggesting that HERP might recruit the proteasome to the ER membrane for efficient ERAD. The aim of this study was to investigate the function of HERP within the UPR. The findings presented here demonstrate that HERP is essential for the degradation of a model ERAD substrate. Thus, HERP indeed has a role in ERAD. Moreover, the data show that HERP directly interacts with the E3 ligase HRD1 and the two proteins form a common protein complex with p97, Derlin-1 and possibly also with VIMP. This suggests that both ubiquitylation and retrotranslocation of ER proteins are performed by one protein complex, enabling an efficient processing of ERAD substrates. This study also demonstrates that the UBL domain of HERP does not share the proteasome binding property of other UBL domains, suggesting that proteasome binding cannot be considered a general feature of all UBL domains. Instead, the HERP UBL domain is able to interact with the deubiquitylating enzyme USP7. Therefore, deubiquitylation might also be an important aspect in the proteasome-dependent degradation of misfolded ER proteins.