Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

Background

Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results

MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion

These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.     

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.     

Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.The encapsidation of the RNA genome is essential for retrovirus replication. Because the viral genomic RNA (gRNA) constitutes only a small fraction of the total cellular mRNA, a specific Gag-RNA interaction is thought to be required for viral genome packaging (2). The determinants of virus-specific gRNA incorporation include the cis-acting element at the 5′end of the viral gRNA, known as the packaging signal (ψ), and the nucleocapsid (NC) domain of the Gag polyprotein (3, 14, 62). In Rous sarcoma virus (RSV), the NC domain contains basic residues that are required for the recognition of and binding to ψ, as well as two Cys-His motifs that maintain the overall conformation of NC and are essential for RNA packaging (30, 31).Packaging of gRNA into progeny virions requires that the unspliced viral mRNA be exported from the nucleus. However, cellular proofreading mechanisms ensure that unspliced or intron-containing mRNAs are retained in the nucleus until splicing occurs. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) overcome this export block of unspliced genomes by encoding the Rev protein, which interacts with a cis-acting sequence in the viral RNA (the Rev-responsive element [RRE]) to facilitate cytoplasmic accumulation of intron-containing viral mRNA (16, 35). The export of the Rev-viral RNA complex is mediated through the interaction of a leucine-rich nuclear export signal (NES) in Rev with the CRM1 nuclear export factor (17, 18, 37, 41). Simple retroviruses do not encode Rev-like regulatory proteins, so other strategies for the export of unspliced viral RNAs are needed. For Mason-Pfizer monkey virus, a cis-acting constitutive transport element induces nuclear export of the unspliced viral RNA in a process mediated by the cellular mRNA nuclear export factor TAP (5, 25, 46, 63). In RSV, an RNA element composed of either of the two direct repeats flanking the src gene mediates the cytoplasmic accumulation of unspliced viral RNA by using host export proteins TAP and Dpb5 (29, 42, 44).The findings of recent studies suggest that specific RNA export pathways direct viral gRNA to sites of virion assembly (56); for example, HIV-1 gRNA export out of the nucleus by the Rev-RRE-CRM1 complex is required for the proper subcellular localization of Gag and efficient virus particle production (26, 57). In the case of RSV, little is known about the trafficking of the viral RNA destined for virion encapsidation or the effects of the gRNA nuclear export pathway on Gag trafficking and virus particle production. However, we do know that RSV Gag enters the nucleus during infection, owing to nuclear localization signals (NLSs) in the matrix (MA) and NC domains. The nuclear localization of Gag is transient, and export is mediated by a CRM1-dependent NES in the p10 region (6, 52, 53). Thus, it is feasible that Gag may facilitate the nuclear export of the gRNA, either directly or indirectly, to promote particle assembly (53).In support of this idea, Gag mutants engineered to be more efficiently directed to the plasma membrane than wild-type Gag by the addition of the Src membrane-binding domain (in Myr1E virus) or by the insertion of extra basic residues (in SuperM virus) are not concentrated in nuclei when cells are treated with the CRM1 inhibitor leptomycin B (LMB) (8, 20, 53). Moreover, Myr1E and SuperM virus particles incorporate reduced levels of viral gRNA compared to the levels incorporated by wild-type particles. Thus, there is a correlation between the nuclear transit of Gag and gRNA packaging, although the Myr1E and SuperM viruses may be deficient in gRNA encapsidation because they are transported to the plasma membrane too rapidly (8). To test the hypothesis that the loss of Gag nuclear trafficking is responsible for the gRNA packaging defect, we used a gain-of-function genetic approach whereby a heterologous NLS was inserted into Myr1E Gag, yielding mutant virus Myr1E.NLS. Our results revealed that restoring the nuclear trafficking of Myr1E Gag also restored the incorporation of gRNA into mutant virus particles.     

RNA Regulatory Elements in the Genomes of Simple Retroviruses

The RNA genomes of simple retroviruses encode three genes (gag, pol,andenv) which are required for replication. In addition, there are at least three well-definedcis-acting structures which regulate important aspects of the viral life cycle. The packaging signal at the 5′ end of the RNA tags the genomic RNA for specific encapsidation into assembling virus. Since viral Env proteins are translated from spliced mRNAs,cis-acting splicing signals ensure that the proper ratio of spliced and unspliced viral RNAs is present in the infected cell. Finally,cis-acting elements at the 3′ end of the genome promote the export of unspliced RNAs from the nucleus for translation and encapsidation.     

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

Background

This study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.

Methodology

Three series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.

Principal Findings

MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.

Conclusions/Significance

These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.     

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 purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77^Gag along with in cell gRNA packaging study identified two Pr77^Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77^Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.     

Translation regulation of mammalian selenoproteins

Background

Interest in selenium research has considerably grown over the last decades owing to the association of selenium deficiencies with an increased risk of several human diseases, including cancers, cardiovascular disorders and infectious diseases. The discovery of a genetically encoded 21^st amino acid, selenocysteine, is a fascinating breakthrough in molecular biology as it is the first addition to the genetic code deciphered in the 1960s. Selenocysteine is a structural and functional analog of cysteine, where selenium replaces sulfur, and its presence is critical for the catalytic activity of selenoproteins.

Dynamic interactions between RNA viruses and human hosts unravelled by a decade of next generation sequencing

Background

Next generation sequencing (NGS) methods have significantly contributed to a paradigm shift in genomic research for nearly a decade now. These methods have been useful in studying the dynamic interactions between RNA viruses and human hosts.

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.

The role of resveratrol and melatonin in the nitric oxide and its oxidation products mediated functional and structural modifications of two glycolytic enzymes: GAPDH and LDH

Background

Nitric oxide is a well-known gaseous signaling molecule and protein modifying agent. However, at higher concentrations or during oxidative stress nitric oxide may exert some deleterious effects on protein structure and function. Here we investigated the influence of nitric oxide and products of its oxidation on two glycolytic enzymes: GAPDH and LDH under in vitro nitrosative stress conditions. Secondly, we applied natural antioxidants: melatonin and resveratrol to examine their effects on the enzymes under studied conditions.