MVH Protein Research Articles

Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific mRNAs in Chromatoid Bodies During Spermatogenesis.

GRTH/DDX25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H) show loss of the phospho-species from cytoplasm with preservation of the non-phospho form in the cytoplasm and nucleus. GRTH KI mice are sterile and lack elongated spermatids and spermatozoa, with spermatogenic arrest at step 8 of round spermatids which contain chromatoid body (CB) markedly reduced in size. We observed an absence of phospho-GRTH in CB of GRTH KI mice. RNA-Seq analysis of mRNA isolated from CB revealed that 1,421 genes show differential abundance, of which 947 genes showed a decrease in abundance and 474 genes showed an increase in abundance in GRTH KI mice. The transcripts related to spermatid development, differentiation, and chromatin remodeling (Tnp1/2, Prm1/2/3, Spem1/2, Tssk 2/3/6, Grth, tAce, and Upf2) were reduced, and the transcripts encoding for factors involved in RNA transport, regulation, and surveillance and transcriptional and translational regulation (Eef1a1, Ppp1cc, Pabpc1, Ybx3, Tent5b, H2al1m, Dctn2, and Dync1h1) were increased in the CB of KI mice and were further validated by qPCR. In the round spermatids of wild-type mice, mRNAs of Tnp2, Prm2, and Grth were abundantly co-localized with MVH protein in the CB, while in GRTH KI mice these were minimally present. In addition, GRTH binding to Tnp1/2, Prm1/2, Grth, and Tssk6 mRNAs was found to be markedly decreased in KI. These results demonstrate the importance of phospho-GRTH in the maintenance of the structure of CB and its role in the storage and stability of germ cell-specific mRNAs during spermiogenesis.

OR02-03 Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific MRNAs in Chromatoid Bodies During Spermiogenesis

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX 25) is a member of the DEAD-box family of RNA helicases which play an essential role in spermatogenesis. There are two species of GRTH, the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in the Japanese azoospermic men which resulted in the lack of phospho-GRTH (pGRTH) in in vitro studies. GRTH knock-in (KI) mice with insertion of the human mutant GRTH gene show loss of the cytoplasmic 61 KDa phospho-species with preservation of the non-phospho nuclear form. KI mice are sterile, lack elongated spermatids and spermatozoa with arrest at step 8 of round spermatids (RS) which contain chromatoid bodies (CB) markedly reduced in size. CB is a non-membranous, cytoplasmic organelle present adjacent to the nucleus of RS, where mRNAs bound to GRTH transported from nucleus to cytoplasmic sites are temporarily stored, translationally repressed for later transport to polyribosomes for translation at specific stages of spermiogenesis. Owing to the specific function of CBs and importance of pGRTH in spermatid elongation, CBs isolated from germ cells of WT and GRTH KI mice were used for subsequent experiments. CBs isolated from GRTH KI mice are smaller, highly condensed and lack the nuage texture of CBs in WT mice. We observed the absence of pGRTH in CB of round spermatids of GRTH KI mice. Also, MVH protein (recognized CB marker protein) was decreased in the CB of GRTH KI mice. Expression of genes related to spermatid regulation, chromatin compaction, remodeling (TP1 and 2, PRM1 and 2, GRTH, TSSK6, HMG2, GCNF, RNF8, TDRD 1, 6, 7 and 9) analyzed by qPCR were markedly reduced in the CB of GRTH KI mice compared to WT. No change was observed in the expression of bromodomain mRNAs and protein, indicating that pGRTH does not participate in the translational regulation of this protein class at the level of this organelle. Notably, mRNAs of TP2, PRM2 and GRTH which associated with GRTH protein were co-localized with MVH protein in the CB. This indicated the relevance of GRTH as a binder/transport protein of key chromatin remodelers for ensuring their mRNA repression/stability within the CB. In addition, GRTH binding to genes essential for spermatid development and regulation (TP1 and 2, PRM1 and 2, GRTH, TSSK6, RNF8 and GCNF) were also found to be markedly decreased in the CB KI mice. These results demonstrate the importance of pGRTH in the maintenance of biochemical composition/structure of the CB and role in spermatid regulation, chromatin compaction, spermatid development and completion of spermatogenesis.

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro.

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated onday 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.

Retargeting of microcell fusion towards recipient cell-oriented transfer of human artificial chromosome.

BackgroundHuman artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. However, the method of introduction of HACs into target cells is confined to microcell-mediated chromosome transfer (MMCT), which is less efficient than other methods of vector introduction. Application of Measles Virus (MV) fusogenic proteins to MMCT instead of polyethylene glycol (PEG) has partly solved this drawback, whereas the tropism of MV fusogenic proteins is restricted to human CD46- or SLAM-positive cells.ResultsHere, we show that retargeting of microcell fusion by adding anti-Transferrin receptor (TfR) single chain antibodies (scFvs) to the extracellular C-terminus of the MV-H protein improves the efficiency of MV-MMCT to human fibroblasts which originally barely express both native MV receptors, and are therefore resistant to MV-MMCT. Efficacy of chimeric fusogenic proteins was evaluated by the evidence that the HAC, tagged with a drug-resistant gene and an EGFP gene, was transferred from CHO donor cells into human fibroblasts. Furthermore, it was demonstrated that no perturbation of either the HAC status or the functions of transgenes was observed on account of retargeted MV-MMCT when another HAC carrying four reprogramming factors (iHAC) was transferred into human fibroblasts.ConclusionsRetargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors. Therefore, this technology could facilitate the systematic cell engineering by HACs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0142-z) contains supplementary material, which is available to authorized users.

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells.

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.

An in vitro study of the impact of 4mT static magnetic field to modify the differentiation rate of rat bone marrow stem cells into primordial germ cells

This investigation was performed to evaluate the differentiation capacity and alteration in genes expression patterns during in vitro differentiation of bone marrow stem cells into primordial germ cells using static magnetic field (4mT) and BMP-4 (25ng/ml). The rate of differentiation was investigated using the Real Time-PCR method for tracing expression of differentiation markers (Oct-4, Nanog, C-Myc, Fragilis, Mvh and Stella). Then, immunocytochemical reaction was carried out for detection of marker proteins (Oct4 and Mvh). Increasing of the exposure time of the 4mT SMF (24 and 48h) and treatment time with 25ng/ml BMP4 (48 and 96h) indicated a marked decrease in expression of pluripotency genes (Oct-4, Nanog and C-Myc) and Oct4 protein and increase in primordial germ cell-specific genes (Fragilis, Mvh and Stella) and Mvh protein compared with the control group. Final results showed that in a synergistic manner, the combination of SMF with BMP4 exaggerates the differentiation potential of BMSCs to PGCs by activating the MAPK pathway and altering the Ca2+ concentration.

Single Cell Analysis Facilitates Staging of Blimp1-Dependent Primordial Germ Cells Derived from Mouse Embryonic Stem Cells

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKitbright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKitbright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5–10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKitbright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

DARPins: An Efficient Targeting Domain for Lentiviral Vectors

We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.

Measles Virus Hemagglutinin: Structural Insights into Cell Entry and Measles Vaccine

Measles is one of the most contagious viral diseases, and remains a major cause of childhood morbidity and mortality worldwide. The measles virus (MV), a member of the family Paramyxoviridae, enters cells through a cellular receptor, the signaling lymphocyte activation molecule (SLAM), CD46 or nectin-4. Entry is mediated by two MV envelope glycoproteins, the hemagglutinin (H) and the fusion (F) protein. The H protein mediates receptor attachment, while the F protein causes membrane fusion. The interaction between the H and F proteins is essential to initiate the cell entry process. Recently determined crystal structures of the MV-H protein unbound and bound to SLAM or CD46 have provided insights into paramyxovirus entry and the effectiveness of measles vaccine.

Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice

Genes of the DAZ family play critical roles in germ cell development in mammals and other animals. In mice, Dazl mRNA is first observed at embryonic day 11.5 (E11.5), but previous studies using Dazl-deficient mice of mixed genetic background have largely emphasized postnatal spermatogenic defects. Using an inbred C57BL/6 background, we show that Dazl is required for embryonic development and survival of XY germ cells. By E14.5, expression of germ cell markers (Mvh, Oct4, Dppa3/Stella, GCNA and MVH protein) was reduced in XY Dazl−/− gonads. By E15.5, most remaining germ cells in XY Dazl−/− embryos exhibited apoptotic morphology, and XY Dazl−/− gonads contained increased numbers of TUNEL-positive cells. The rare XY Dazl−/− germ cells that persisted until birth maintained a nuclear morphology that resembled that of wildtype germ cells at E12.5–E13.5, a critical developmental period when XY germ cells lose pluripotency and commit to a spermatogonial fate. We propose that Dazl is required as early as E12.5–E13.5, shortly after its expression is first detected, and that inbred Dazl−/− mice of C57BL/6 background provide a reproducible standard for exploring Dazl's roles in embryonic germ cell development.

Dazl binds in vivo to specific transcripts and can regulate the pre-meiotic translation of Mvh in germ cells

Gametogenesis is a complex process subject to strict controls at both levels of transcription and translation. Members of a family of conserved RNA-binding proteins encoded by the DAZ genes are required for the translational regulation of gene expression essential for this process. Although loss of DAZ family genes is associated with infertility in several organisms including humans, the identity of the transcripts regulated in vivo is unknown. Using a combination of immunoprecipitation and microarray analysis, we have identified a number of mRNAs that are bound by the murine Dazl protein both in vivo and in vitro. Sequence analysis shows that these transcripts contain binding sites for Dazl, which have been conserved during evolution between human, rat and mouse. We have focussed on mouse vasa homologue (Mvh), a gene that is essential for male gametogenesis, and show that Dazl stimulates translation via the Mvh 3'-UTR. Finally, we show that germ cells of Dazl null mice contain reduced levels of Mvh protein, indicating that Dazl-mediated regulation of Mvh translation is crucial for mammalian spermatogenesis.

Mouse RanBPM is a partner gene to a germline specific RNA helicase, mouse vasa homolog protein.

Germ cell-specific ATP-dependent RNA helicase, the product of the mouse vasa homolog (Mvh), has been shown to play an essential role in the development of the male germ cell. In male Mvh knockout mice, premeiotic germ cells arrest at the zygotene stage. To investigate the role of MVH protein in the progression of meiosis, we searched for genes encoding partners that interact with MVH in testicular germ cells. Using the yeast two-hybrid system, we found that MVH interacts with mouse RanBPM, a Ran-GTP binding protein involved in microtubule nucleation. RanBPM is predominantly expressed in the testis, especially in maturating spermatocytes. Within the cell, RanBPM and MVH are closely associated with perinuclear RNA-protein complexes and chromatoid bodies. The interaction of MVH with RanBPM points to a functional relationship between translational regulation and the microtubule nucleation during meiosis. Mol. Reprod. Dev. 66: 1-7, 2004.

Distinct Kinetics for Binding of the CD46 and SLAM Receptors to Overlapping Sites in the Measles Virus Hemagglutinin Protein

Measles virus (MV) is a human pathogen using two distinct cell surface receptors for entry into host cells. We present here a comparative analysis for binding of the MV receptors CD46 and SLAM to the measles virus hemagglutinin protein (MVH, Edmonston strain). Soluble monomeric and dimeric MVH variants were prepared in mammalian cells and their conformation assessed using a panel of monoclonal antibodies. The two receptor molecules specifically bound to the MVH protein with distinct binding modes. The association rate (k(a)) for SLAM binding to MVH was very low ( approximately 3000 m(-1)s(-1)), about 20 times lower that the k(a) determined for CD46 binding. However, SLAM bound tighter to the virus protein than CD46, as revealed by a 5-fold lower dissociation rate (k(d), approximately 1.5 x 10(-3) s(-1)). These data suggest that the SLAM receptor binds to a less accessible and more hydrophobic surface on MVH than the CD46 receptor, as illustrated in a binding model. Despite the differences in kinetics, receptor competition binding experiments revealed that they recognize overlapping sites in MVH. Indeed, a panel of anti-MVH monoclonal antibodies equally inhibited binding of both receptor molecules. The similar immune reactivity of the two receptor binding sites suggests that the shift in receptor usage by MV may not be driven by immune responses.

Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of Peste des petits ruminants virus: identification of a T cell determinant

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus within the family Paramyxoviridae, causes a fatal disease ‘peste des petits ruminants ’ in goats and sheep. This enveloped virus is antigenically closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants. PPRV harbors two major surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. The surface glycoproteins of morbilliviruses are highly immunogenic and confer protective immunity. In this study, we investigated the immune responses generated in goats immunized with low doses of purified recombinant extracellular baculovirus carrying a membrane bound form of the HN protein of PPRV without any adjuvant. We report that the immunized goats develop both humoral and cell-mediated immune responses. Antibodies generated in the immunized animals could neutralize both PPRV and RPV in vitro. Further, using a combination of Escherichia coli expressed deletion mutants of PPRV-HN and RPV-H proteins, and synthetic peptides corresponding to the highly conserved N-terminal sequences of MV-H protein, we have mapped an N-terminal T cell determinant (amino acids 123–137) and a C-terminal domain (amino acids 242–609) harboring potential T cell determinant(s) in goats.

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.

A simplified immunoassay based on measles virus recombinant hemagglutinin protein for testing the immune status of vaccinees

Simplified tests based on recombinant antigens are considered to be important for monitoring immunity against measles virus (MV). The hemagglutinin protein (H) is the main target for neutralising and protective antibodies. We produced a recombinant MV-H protein, in a high-yield mammalian expression system based on the Semliki Forest virus replicon. The antigenicity of this recombinant protein was investigated with monoclonal antibodies and its suitability for measuring the immune status of vaccinees was tested in a large cohort by ELISA (H-ELISA). The results were evaluated against neutralisation (NT) and hemagglutination inhibition (HI) titers and MV-specific IgG measured in a commercial whole-virus based ELISA (MV-ELISA, Enzygnost). The H-ELISA correlated better with HI ( r=0.78) and NT titers ( r=0.80), than the MV-ELISA (HI, r=0.58; NT, r=0.59). In contrast to the MV-ELISA, the H-ELISA detected no false-positive sera ( P<0.02) and the number of false-negative sera was significantly lower in the H-ELISA than in the MV-ELISA (4/378 vs. 15/378; P<0.025). The performance of the H-ELISA did not deteriorate significantly when, instead of background corrected net values, uncorrected raw O.D. values of the H-antigen were considered, or when early time points (30 min) were evaluated. These results demonstrate that the recombinant H-ELISA detects efficiently non-immune individuals among vaccinees, despite their relatively low MV-antibody levels. A simplified format with single value measurements did not result in loss of sensitivity or specificity and its performance compared favorably with commercial ELISAs based on whole virus.

Cell-to-cell contact via measles virus haemagglutinin-CD46 interaction triggers CD46 downregulation

CD46 downregulation by measles virus (MV) occurs after expression of virus haemagglutinin (H) protein on the surface of the infected cell and is a consequence of CD46-H interaction on the cell membrane. To assess whether CD46 downregulation also occurs after CD46-H interaction when these two molecules are expressed on distinct cells, we used human T cell line Jurkat (expressing CD46) and transfected murine fibroblast line L stably expressing MV-H protein (L.H). FACS analysis shows that cell-to-cell contact of 1 h at 37 degrees C triggers a reduction of CD46 cell surface labelling as detected by MCI20.6, GB24 and J4-48 monoclonal antibodies. This reduction is similar to that observed after MV infection or after infection with recombinant vaccinia virus encoding MV-H protein. By contrast, MV-H protein was downregulated only when CD46-H interaction occurred on the same cell membrane. CD46 downregulation is specific for CD46-H interaction because it was not observed after coincubation of Jurkat cells with either L cells expressing MV nucleoprotein (L.NP) or L cells. Moreover, this downregulation could be blocked by either anti-CD46 or anti-H antibodies. The H-mediated CD46 downregulation is reversible and restricted to CD46 since expression of other surface markers (CD3, CD14, CD47 and CD63) is unaffected. It is apparently not mediated in a protein kinase (PK) A- or PKC-dependent manner. Altogether, our results provide an unequivocal demonstration that interaction between the extracellular domains of CD46 and MV-H is sufficient to trigger CD46 downregulation.

Molecular cloning and sequence analysis of the rinderpest virus mRNA encoding the hemagglutinin protein

We cloned the full-length cDNAs corresponding to the mRNA for the hemagglutinin[dH) protein of rinderpest virus (RV) and determined the nucleotide sequence of RV-H. The gene of RV-H was composed of 1952 nucleotides and contained a single large open reading frame, which was capable of encoding a protein of 609 amino acids with a molecular weight of 68,330 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of the measles virus (MV)-H. The 5′ end of the message(nucleotides 1 to 485) was largely conserved, with a homology of 75.1% of the nucleotides and 78.0% of the predicted amino acids. In the middle portion[dnucleotides 486–1310), where the potential glycosylation sites exist, 56.6% of the nucleotides and 49.5% of the amino acids were identical. In the 3′ end of the message[dnucleotides 1311–1850), 63.3% of the nucleotides and 58.1% of the amino acids were identical. Four potential glycosylation sites were found in RV-H protein and three of them were the same as those of MV-H protein. The positions of 13 cysteine residues of RV-H were absolutely identical to those of MV-H. The hydropathy profile of RV-H protein resembled that of MV-H. One major hydrophobic region long enough to be an anchor in the membrane was located near the N-terminus.