Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element

Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3′ long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3′ long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3′-end of the mouse mammary tumor virus genome, but further deletions at the 5′- or 3′-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3′-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5′- and 3′-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure.Mouse mammary tumor virus (MMTV)³ has multiple regulatory and accessory genes (1, 2). The known accessory genes specify a dUTPase (3), which is believed to be involved in retroviral replication in non-dividing cells (4), as well as superantigen (Sag). Sag is a transmembrane glycoprotein that is involved in the lymphocyte-mediated transmission of MMTV from maternal milk in the gut to susceptible epithelial cells in the mammary gland (5, 6). The Sag protein expressed by endogenous (germline) MMTV proviruses has been reported to provide susceptibility to infection by exogenous MMTVs or the bacterial pathogen, Vibrio cholerae (7). These results suggest a role for MMTV Sag in the host innate immune response.MMTV recently was shown to be a complex retrovirus (1). Complex retroviruses encode RNA-binding proteins that facilitate nuclear export of unspliced viral RNA by using a leucine-rich nuclear export sequence (8), which binds to chromosome region maintenance 1 (Crm1)(9), whereas simple retroviruses have a cis-acting constitutive transport element that directly interacts with components of the Tap/NXF1 pathway (10). Similar to other complex retroviruses, MMTV encodes a Rev-like protein, regulator of export/expression of MMTV mRNA (Rem) (1). Rem is translated from a doubly spliced mRNA into a 33-kDa protein that contains nuclear and nucleolar localization signals as well as a predicted RNA-binding motif and leucine-rich nuclear export sequence (1, 2). Our previous experiments indicated that Rem affects export of unspliced viral RNA, and a reporter vector that relies on luciferase expression from unspliced RNAs has increased activity in the presence of Rem (1). Sequences at the MMTV envelope-long terminal repeat (LTR) junction were required within the vector for Rem-induced expression, suggesting that the LTR contains all or part of the Rem-responsive element (RmRE). Very recently, Müllner et al. (11) identified a 490-nt region spanning the MMTV envelope-3′ LTR region, which was predicted to form a highly structured RNA element. This element confers Rem responsiveness on heterologous human immunodeficiency virus type 1 (HIV-1)-based plasmid constructs in transfection experiments.Experiments using other retroviral export proteins have demonstrated considerable variation in the size of the response elements. A minimal Rev-responsive element (RRE) in the human immunodeficiency virus type 1 (HIV-1) genomic RNA is 234 nt, the human T-cell leukemia virus Rex-responsive element is 205 nt (12–14), whereas the Rec-responsive element (RcRE; also known as the K-RRE) of human endogenous retrovirus type K is 416 to 429 nt (15, 16). Most response elements are confined to the 3′-end of their respective retroviral genomes (either to the envelope or LTR regions) (14, 15), but 5′ Rev-response elements also have been identified (17). Studies indicate that the secondary structure is a critical factor for proper function of retroviral response elements (18), and that multiple stem-loops are required. Export proteins multimerize on these elements to allow activity (19).In the current study, we have used deletion mutations within a reporter vector based on the 3′-end of the MMTV genome to define a 476-nt element necessary for maximum Rem responsiveness. This element spans the envelope-LTR junction of the MMTV genome as previously reported (1). However, a secondary structure model generated using digestions of the RmRE by RNases V1, T1, and A as experimental constraints differs significantly from the published structure (11) and more closely resembles complex retroviral response elements. Transfection experiments indicated that the MMTV RmRE could function in both mouse and human cells, suggesting that conserved cellular proteins interact with Rem.     

Changes in microRNA expression profiles in HIV-1-transfected human cells

Background

The HIV-1 Rev regulatory protein binds as an oligomeric complex to viral RNA mediating nuclear export of incompletely spliced and non-spliced viral mRNAs encoding the viral structural proteins. However, the biological significance of the obligatory complex formation of Rev upon the viral RNA is unclear.

Results

The activity of various fusion proteins based on the negative oligomerization-defect Rev mutant M4 was tested using Rev dependent reporter constructs. An artificial M4 mutant dimer and an M4 mutant containing an extra basic domain from the HTLV-I Rex protein exhibited nearly full activity when compared to wild type Rev.

Conclusion

Rev dimerization appears to be required to expose free basic domains whilst the Rev oligomeric complex remains bound to viral RNA via other basic domains.     

Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4

Background

The human endogenous retrovirus HERV-K(HML-2) family is associated with testicular germ cell tumors (GCT). Various HML-2 proviruses encode viral proteins such as Env and Rec.

Results

We describe here that HML-2 Env gives rise to a 13 kDa signal peptide (SP) that harbors a different C-terminus compared to Rec. Subsequent to guiding Env to the endoplasmatic reticulum (ER), HML-2 SP is released into the cytosol. Biochemical analysis and confocal microscopy demonstrated that similar to Rec, SP efficiently translocates to the granular component of nucleoli. Unlike Rec, SP does not shuttle between nucleus and cytoplasm. SP is less stable than Rec as it is subjected to proteasomal degradation. Moreover, SP lacks export activity towards HML-2 genomic RNA, the main function of Rec in the original viral context, and SP does not interfere with Rec's RNA export activity.

Conclusion

SP is a previously unrecognized HML-2 protein that, besides targeting and translocation of Env into the ER lumen, may exert biological functions distinct from Rec. HML-2 SP represents another functional similarity with the closely related Mouse Mammary Tumor Virus that encodes an Env-derived SP named p14. Our findings furthermore support the emerging concept of bioactive SPs as a conserved retroviral strategy to modulate their host cell environment, evidenced here by a "retroviral fossil". While the specific role of HML-2 SP remains to be elucidated in the context of human biology, we speculate that it may be involved in immune evasion of GCT cells or tumorigenesis.     

Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm.

All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for maintenance of this RNA in the cytoplasm, where it is either translated to produce Gag and Pol proteins or packaged into viral particles. The complex retroviruses encode Rev or Rex regulatory proteins, which interact with cis-acting viral sequences to promote cytoplasmic expression of incompletely spliced viral RNAs. Since the simple retroviruses do not encode regulatory proteins, we proposed that they might contain cis-acting sequences that could interact with cellular Rev-like proteins. To test this possibility, we initially looked for a cis-acting sequence in avian retroviruses that could substitute for Rev and the Rev response element in human immunodeficiency virus type 1 expression constructs. A cis-acting element in the 3' untranslated region of Rous sarcoma virus (RSV) RNA was found to promote Rev-independent expression of human immunodeficiency virus type 1 Gag proteins. This element was mapped between RSV nucleotides 8770 and 8925 and includes one copy of the direct repeat (DR) sequences flanking the RSV src gene; similar activity was observed for the upstream DR. To address the function of this element in RSV, both copies of the DR sequence were deleted. Subsequently, each DR sequence was inserted separately back into this deleted construct. While the viral construct lacking both DR sequences failed to replicate, constructs containing either the upstream or downstream DR replicated well. In the absence of both DRs, Gag protein levels were severely diminished and cytoplasmic levels of unspliced viral RNA were significantly reduced; replacement of either DR sequence led to normal levels of Gag protein and cytoplasmic unspliced RNA.     

Identification and Mutational Analysis of a Rej Response Element in Jaagsiekte Sheep Retrovirus RNA

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5′ end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3′ end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus that causes ovine pulmonary adenocarcinoma, an infectious lung tumor of sheep (10, 29). Ovine pulmonary adenocarcinoma has morphological resemblance to a human lung cancer, bronchioloalveolar carcinoma, which is only weakly associated with cigarette smoking. In recent years, complete infectious and oncogenic molecular clones of JSRV have been isolated (30). We and others found that the JSRV envelope (Env) protein also functions as an oncogene in that it can induce morphological transformation of fibroblast and epithelial cell lines in culture and tumors in animals (1, 24, 34). Further studies have demonstrated that amino acids in the cytoplasmic tail of the Env transmembrane (TM) protein are important for transformation, as are multiple domains in the surface (SU) protein (17, 18).The nuclear export of mRNA is a critical step in gene expression. All retroviruses employ unspliced genome-length RNA as mRNA for synthesis of Gag and Pol proteins, while splicing yields mRNA(s) for Env (and other) proteins (15). Thus, genome-length mRNA for Gag and Pol is equivalent to an unspliced precursor for Env mRNA. A key issue for retroviruses is how they transport unspliced genome-length RNA to the cytoplasm. This is accomplished by two general mechanisms. The human immunodeficiency virus type 1 (HIV-1) Rev protein (encoded by a doubly spliced mRNA) specifically binds to a Rev-responsive element (RRE), located in RNA of the env gene. The Rev/RRE complex recruits the cellular CRM1/Xpo1 protein (as well as other cellular proteins), which results in transport of this RNA-protein complex to the cytoplasm (7). Similarly, human T-cell leukemia virus type 1 (HTLV-1) Rex protein binds a Rex-responsive element on viral RNA, resulting in export via the CRM1 pathway (21). The betaretroviruses mouse mammary tumor virus (MMTV) and human endogenous retrovirus K (HERV-K) also encode analogous regulatory proteins (Rem and Rec, respectively) (19, 22, 27).In contrast, the betaretroviruses Mason-Pfizer monkey virus (MPMV) and simian retrovirus (SRV) contain constitutive RNA export elements (constitutive transport elements [CTEs]) that facilitate nuclear export of unspliced RNA (4, 41). The MPMV CTE is located between env and the 3′ long terminal repeat (LTR); it binds to the cellular trans-acting factor NXF1/Tap, which directs nuclear export of the RNA-protein complex to the cytoplasm (14). Rous sarcoma virus and the related avian leukosis viruses contain direct repeat sequences flanking the src gene or in the 3′ untranslated region of their RNA (28). Structure-function analyses of these RNA-exporting elements revealed specific stem-loop structures that are important for activity and for binding of the host cell factors (3).Like other betaretroviruses, JSRV contains the standard genes gag, pro, pol, and env. In addition we recently found that JSRV also encodes a regulatory factor, Rej (17a). Rej is reminiscent of MMTV Rem and HERV-K Rec in that it is encoded in the 5′ end of env and it is required for efficient synthesis of Gag protein. We found that Rej is required for translation of unspliced viral RNA, and in 293T cells it also enhances accumulation of cytoplasmic unspliced viral RNA in the cytoplasm. In the results presented here, we show that JSRV RNA also contains a Rej-responsive element (RejRE) in the 3′ end of env that is required for translation of Gag protein and efficient export or accumulation of unspliced viral RNA in the cytoplasm in 293T cells. Mutational analyses of RejRE based on M-fold suggest that both primary sequences and secondary structures in this region play important roles in nuclear export or accumulation of unspliced viral RNA in the cytoplasm and Gag synthesis. This accumulation is independent of Tap but dependent on CRM1. Moreover, Rej protein was exported from the nucleus to the cytoplasm in cells expressing wild-type JSRV RNA but not RejRE mutants, suggesting binding of Rej protein to the RejRE.     

A nuclear kinesin-like protein interacts with and stimulates the activity of the leucine-rich nuclear export signal of the human immunodeficiency virus type 1 rev protein

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.     

A rev-like NES mediates cytoplasmic localization of HERV-K cORF

The human endogenous retrovirus K (HERV-K)-encoded protein cORF has recently been shown to be a functional homolog of the HIV Rev protein. Rev-mediated RNA export requires interaction between a leucine-rich nuclear export signal (NES) in Rev and the nuclear export receptor Crm1/exportin1. Like Rev, cORF binds to Crm1 and cORF-mediated RNA export depends on Crm1 activity. Here we document that mutation of the putative NES in cORF results in trapping of the protein in the nucleus, suggesting that the cORF NES functions in analogy to the Rev NES.     

A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.     

Frequency of Human Endogenous Retroviral Sequences (HERV) K113 and K115 in the Polish Population,and Their Effect on HIV Infection

Background

The human genome contains about 8% of endogenous retroviral sequences originated from germ cell infections by exogenous retroviruses during evolution. Most of those sequences are inactive because of accumulation of mutations but some of them are still capable to be transcribed and translated. The latter are insertionally polymorphic HERV-K113 and HERV-K115. It has been suggested that their presence and expression was connected with several human diseases. It is also believed that they could interfere with the replication cycle of exogenous retroviruses, including HIV.

Results

Prevalence of endogenous retroviral sequences HERV-K113 and HERV-K115 was determined in the Polish population. The frequencies were found as 11.8% for HERV-K113 and 7.92% for HERV-K115. To verify the hypothesis that the presence of these HERVs sequences could affect susceptibility to HIV infection, comparison of a control group (HIV-negative, not exposed to HIV; n = 303) with HIV-positive patients (n = 470) and exposed but uninfected (EU) individuals (n = 121) was performed. Prevalence of HERV-K113 and HERV-K115 in the EU group was 8.26% and 5.71%, respectively. In the HIV(+) group we detected HERV-K113 sequences in 12.98% of the individuals and HERV-K115 sequences in 7.23% of the individuals. There were no statistically significant differences between groups studied.

Conclusion

The frequency of HERV-K113 and HERV-K115 sequences in Poland were found to be higher than usually shown for European populations. No relation between presence of the HERVs and HIV infection was detected.     

Regulation of human T cell leukemia virus expression

Retroviruses of the type C morphology have been implicated in a wide variety of diseases in animals and humans. The human T cell leukemia viruses types I (HTLV-I) and II (HTLV-II), the prototypic human-type C retroviruses, have been identified as the causative agents of some forms of human leukemia and neurological disorders. The genetic structure and regulation of the HTLVs are more complex than their avian and murine leukemia virus counterparts. In addition to the gag, pol, and env genes that encode the characteristic virion proteins of all replication competent retroviruses, the genomes of HTLV encode the non-structural proteins, Tax and Rex, which are required for regulating viral gene expression. To understand what appears to be a complex mechanism of disease induction by HTLV, elucidating the regulation and function of the viral gene products and the interaction of these products with each other, as well as with cellular factors, will be critical. This review focuses primarily on regulation of HTLV gene expression in the infected human T lymphocyte, but also discusses analogous gene regulation by the human immunodeficiency virus (HIV). It concentrates specifically on the role these gene products play in virus replication and, ultimately, pathogenesis.