Enzootic nasal tumor virus envelope requires a very acidic pH for fusion activation and infection

Enzootic nasal tumor virus (ENTV) is a close relative of jaagsiekte sheep retrovirus (JSRV), and the two viruses use the same receptor, hyaluronidase 2 (Hyal2), for cell entry. We report here that, unlike the JSRV envelope (Env) protein, the ENTV Env protein does not induce cell fusion at pHs of 5.0 and above but requires a much lower pH (4.0 to 4.5) for fusion to occur. The entry of ENTV Env pseudovirions was substantially inhibited by bafilomycin A1 (BafA1) but was surprisingly enhanced by lysosomotropic agents and lysosomal protease inhibitors following a 4- to 6-h treatment period; of note, prolonged treatment with BafA1 or ammonium chloride completely blocked ENTV entry. Unlike typical pH-dependent viruses, ENTV Env pseudovirions were virtually resistant to inactivation at a low pH (4.5 or 5.0). Using chimeras formed from ENTV and JSRV Env proteins, we demonstrated that the transmembrane (TM) subunit of ENTV Env is primarily responsible for its unusually low pH requirement for fusion but found that the surface (SU) subunit of ENTV Env also critically influences its relatively low and pH-dependent fusion activity. Furthermore, the poor infectivity of ENTV pseudovirions in human cells was significantly improved by either replacing the SU subunit of ENTV Env with that of JSRV Env or overexpressing the functional Hyal2 receptor in target cells, suggesting that ENTV SU-Hyal2 interaction is likely to be the limiting step for viral infectivity. Collectively, our data reveal that the fusogenicity of ENTV Env is intrinsically lower than that of JSRV Env and that ENTV requires a more acidic pH for fusion, which may occur in an intracellular compartment(s) distinct from that used by JSRV.     

Murine leukemia virus R Peptide inhibits influenza virus hemagglutinin-induced membrane fusion

The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.     

Human T-cell leukemia virus type 1 envelope-mediated syncytium formation can be activated in resistant Mammalian cell lines by a carboxy-terminal truncation of the envelope cytoplasmic domain

Human T-cell leukemia virus (HTLV) envelope (Env) glycoproteins induce fusion, leading to rampant syncytium formation in a broad range of cell lines. Here, we identified murine, hamster, canine, and porcine cell lines that are resistant to HTLV-1 Env-induced syncytium formation. This resistance was not due to the absence of functional receptors for HTLV Env, as these cells were susceptible to infection with HTLV Env-pseudotyped virions. As murine leukemia virus (MLV) Env and HTLV Env present close structural homologies (F. J. Kim, I. Seiliez, C. Denesvre, D. Lavillette, F. L. Cosset, and M. Sitbon, J. Biol. Chem. 275:23417-23420, 2000), and because activation of syncytium formation by MLV Env generally requires cleavage of the R peptide in the cytoplasmic domain of the Env transmembrane (TM) component, we assessed whether truncation of the cytoplasmic domain of HTLV Env would alleviate this resistance. Indeed, in all resistant cell lines, truncation of the last 8 amino acids of the HTLV Env cytoplasmic domain (HdC8) was sufficient to overcome resistance to HTLV Env-induced syncytium formation. Furthermore, HdC8-mediated cell-to-cell infection titers varied according to the target cell lines and could be significantly higher than that observed with HTLV Env on HeLa cells. These data indicate that a determinant located within the 8 carboxy-terminal cytoplasmic amino acids of TM plays a distinct role in HTLV Env-mediated cell-to-cell infection and syncytium formation.     

Receptor Binding and Low pH Coactivate Oncogenic Retrovirus Envelope-Mediated Fusion

Fusion of enveloped viruses with host cells is triggered by either receptor binding or low pH but rarely requires both except for avian sarcoma leukosis virus (ASLV). We recently reported that membrane fusion mediated by an oncogenic Jaagsiekte sheep retrovirus (JSRV) envelope (Env) requires an acidic pH, yet receptor overexpression is required for this process to occur. Here we show that a soluble form of the JSRV receptor, sHyal2, promoted JSRV Env-mediated fusion at a low pH in normally fusion-negative cells and that this effect was blocked by a synthetic peptide analogous to the C-terminal heptad repeat of JSRV Env. In contrast to the receptor of ASLV, sHyal2 induced pronounced shedding of the JSRV surface subunit, as well as unstable conformational rearrangement of its transmembrane (TM) subunit, yet full activation of JSRV Env fusogenicity, associated with strong TM oligomerization, required both sHyal2 and low pH. Consistently, sHyal2 enabled transduction of nonpermissive cells by JSRV Env pseudovirions, with low efficiency, but substantially blocked viral entry into permissive cells at both binding and postbinding steps, indicating that sHyal2 prematurely activates JSRV Env-mediated fusion. Altogether, our study supports a model that receptor priming promotes fusion activation of JSRV Env at a low pH, and that the underlying mechanism is likely to be different from that of ASLV. Thus, JSRV may provide a useful alternate model for the better understanding of virus fusion and cell entry.Fusion is a fundamental event in the life cycle of enveloped viruses and is essential for viral replication. While viral fusion proteins are highly divergent in primary sequence, their structures and modes of activation share striking similarities, permitting their classification into two major groups (41). Class I fusion proteins, as exemplified by the retrovirus envelope (Env) and influenza virus hemagglutinin (HA), are composed mainly of alpha-helices, and they are present as metastable trimers on the viral surface (11). Class II fusion proteins, represented by alphavirus E1 and flavivirus E, contain predominantly beta-sheets and exist as dimers in the prefusion state (16). Of note, the vesicular stomatitis virus G (VSV-G) and herpesvirus gB proteins were recently assigned to a newly established class III, for fusion proteins combining properties of both class I and class II (13, 30). Despite these differences, one common and intriguing characteristic of all viral fusion proteins is their ability to undergo dramatic conformational rearrangements upon activation, i.e., the formation of trimers of hairpins, which drive fusion between viral and cellular membranes (11, 17).Retrovirus Env is a typical type I transmembrane protein composed of surface (SU) and transmembrane (TM) subunits and belongs to the class I fusion proteins. SU is responsible for binding to cognate cellular receptors or cofactors, while TM directly mediates membrane fusion (6). Most retroviruses use a pH-independent pathway for entry, during which receptor binding relieves the ability of SU to restrain TM, resulting in conformational changes in TM and subsequent fusion with the cell membrane (11). Interestingly, increasing numbers of retroviruses have recently been shown to require a low pH (3, 15, 24, 28, 31) or pH-dependent protease activities to trigger fusion (18); the latter property has also been demonstrated for some other enveloped viruses (2, 14, 18, 26, 27, 33, 34). Among these, avian sarcoma leukosis virus (ASLV) is unique in that it uses a two-step mechanism for fusion, in which receptor binding primes the second trigger of low pH (24).Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus etiologically responsible for contagious lung tumors in sheep (12). The native Env protein of JSRV functions as a potent oncogene that induces cell transformation in vitro and in animals (4, 9, 21, 29, 42). The cell entry receptor for JSRV has been identified as hyaluronidase 2 (Hyal2), a glycosylphosphatidylinositol (GPI)-anchored protein belonging to the hyaluronidase family (29); Hyal2 itself has low hyaluronidase activity, and this activity is not associated with JSRV entry and infection (38). Intrigued by the oncogenic nature of JSRV Env, we recently examined the mechanism of JSRV entry and found that JSRV Env-mediated fusion and cell entry require a low pH (1, 8). These observations led us to hypothesize that the pH-dependent fusion activation of JSRV Env may be advantageous for its oncogenesis, given that extreme cell-cell fusion of the plasma membrane at a neutral pH would result in syncytium formation and often cell death. Curiously, we noticed that overexpression of Hyal2 is necessary for JSRV Env to induce membrane fusion at a low pH in vitro, suggesting that Hyal2 may play an active role in the pH-dependent fusion process. Here we provide direct evidence that Hyal2 functions in cooperation with a low pH to trigger the JSRV Env-mediated fusion activation yet exhibits some striking differences from the mechanism of ASLV fusion. The multistep pathway for JSRV Env-mediated fusion activation might be important for its replication fitness and oncogenesis.     

Mutations in the cytoplasmic tail of murine leukemia virus envelope protein suppress fusion inhibition by R peptide

During viral maturation, the cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein undergoes proteolytic cleavage by the viral protease to release the 16-amino-acid R peptide, and this cleavage event activates the Env protein's fusion activity. We introduced Gly and/or Ser residues at different positions upstream of the R peptide in the cytoplasmic tail of the Friend MuLV Env protein and investigated their effects on fusion activity. Expression in HeLa T4 cells of a mutant Env protein with a single Gly insertion after I619, five amino acids upstream from the R peptide, induced syncytium formation with overlaid XC cells. Env proteins containing single or double Gly-Ser insertions after F614, 10 amino acids upstream from the R peptide, induced syncytium formation, and mutant proteins with multiple Gly insertions induced various levels of syncytium formation between HeLa T4 and XC cells. Immunoprecipitation and surface biotinylation assays showed that most of the mutants had surface expression levels comparable to those of the wild-type or R peptide-truncated Env proteins. Fluorescence dye redistribution assays also showed no hemifusion in the Env proteins which did not induce fusion. Our results indicate that insertion mutations in the cytoplasmic tail of the MuLV Env protein can suppress the inhibitory effect of the R peptide on membrane fusion and that there are differences in the effects of insertions in two regions in the cytoplasmic tail upstream of the R peptide.     

Membrane fusion and cell entry of XMRV are pH-independent and modulated by the envelope glycoprotein's cytoplasmic tail

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry.     

Low pH is required for avian sarcoma and leukosis virus Env-induced hemifusion and fusion pore formation but not for pore growth

Binding of avian sarcoma and leukosis virus (ASLV) to its cognate receptor on the cell surface causes conformational changes in its envelope protein (Env). It is currently debated whether low pH is required for ASLV infection. To elucidate the role of low pH, we studied the association between ASLV subgroup B (ASLV-B) and liposomes and fusion between effector cells expressing Env from ASLV-A and ASLV-B and target cells expressing cognate receptors. Neither EnvA nor EnvB promoted cell-cell fusion at neutral pH, but lowering the pH resulted in quick and extensive fusion. As expected for a low-pH-triggered reaction, fusion was a steep function of pH. Steps that required low pH were identified. Binding a soluble form of the receptor caused ASLV-B to hydrophobically associate with liposome membranes at neutral pH, indicating that low pH is not required for insertion of Env's fusion peptides into membranes. But both cell-cell hemifusion and fusion pore formation were pH dependent. It is proposed that fusion peptide insertion stabilizes the conformation of ASLV Env into a form that can be acted upon by low pH. At this point, but not before, low pH can induce fusion and is in fact required for fusion to occur. However, low pH is no longer necessary after formation of the initial fusion pore: pore enlargement does not require low pH.     

Critical role of leucine-valine change in distinct low pH requirements for membrane fusion between two related retrovirus envelopes

Many viruses use a pH-dependent pathway for fusion with host cell membrane, the mechanism of which is still poorly understood. Here we report that a subtle leucine (Leu)-valine (Val) change at position 501 in the envelope glycoproteins (Envs) of two related retroviruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), is responsible for their distinct low pH requirements for membrane fusion and infection. The Leu and Val residues are predicted to reside within the C-terminal heptad repeat (HR2) region of JSRV and ENTV Envs, particularly proximal to the hairpin turn of the putative six-helix bundle (6HB). Substitution of the JSRV Leu with a Val blocked the Env-mediated membrane fusion at pH 5.0, whereas replacement of the ENTV Val with a Leu rendered the ENTV Env capable of fusing at pH 5.0. A Leu-Val change has no apparent effect on the stability of native Env, but appears to stabilize an intermediate induced by receptor binding. These results are consistent with the existence of at least two metastable conformations of these viral glycoproteins, the native prefusion conformation and a receptor-induced metastable intermediate. Collectively, this work represents an interesting perhaps unique example whereby a simple Leu-Val change has critical impact on pH-dependent virus fusion and entry.     

Multiple domains of the Jaagsiekte sheep retrovirus envelope protein are required for transformation of rodent fibroblasts

Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma. We previously showed that the gene encoding JSRV envelope protein (Env) appears to function as an oncogene, since it can transform mouse NIH 3T3 cells. The cytoplasmic tail of the Env transmembrane protein (TM) is necessary for the transformation. However, previous experiments did not exclude the involvement of the Env surface protein (SU) in transformation. In this study, we created a series of nested deletion mutants through the SU domain and assessed their ability to transform rodent fibroblasts. All SU deletion mutants downstream of the predicted signal peptide were unable to transform murine NIH 3T3 or rat 208F cells. Transport to the plasma membrane of selected deleted Env proteins was confirmed by confocal immunofluorescence microscopy of hemagglutinin-tagged versions. Additional sequential SU deletion mutants lacking 50-amino-acid (aa) blocks throughout SU also were unable to transform. Furthermore, minimal insertion mutants of two amino acids (Leu/Gln) at various positions in SU also abolished transformation. These data indicate that domains in SU facilitate efficient JSRV transformation. This could reflect a necessity of SU for appropriate configuration of the Env protein or independent activation by SU of a signaling pathway necessary for transformation. Complementation between SU and TM mutants for transformation supported the latter hypothesis. Cotransfection with DeltaGP Y590F (mutant in the TM cytoplasmic tail) with DeltaGP SUDelta103-352 (lacking most of SU) resulted in efficient transformation. The resulting transformants showed evidence for the presence and expression of both mutant plasmids.     

Influence of acylation sites of influenza B virus hemagglutinin on fusion pore formation and dilation

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.     

Role of the cytoplasmic tail of ecotropic moloney murine leukemia virus Env protein in fusion pore formation

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4 degrees C and the time until fusion measured after the temperature was raised to 37 degrees C or whether cells were first brought into contact at 37 degrees C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.     

Residues of the human metapneumovirus fusion (F) protein critical for its strain-related fusion phenotype: implications for the virus replication cycle

The paramyxovirus F protein promotes fusion of the viral and cell membranes for virus entry, as well as cell-cell fusion for syncytium formation. Most paramyxovirus F proteins are triggered at neutral pH to initiate membrane fusion. Previous studies, however, demonstrated that human metapneumovirus (hMPV) F proteins are triggered at neutral or acidic pH in transfected cells, depending on the strain origin of the F sequences (S. Herfst et al., J. Virol. 82:8891-8895, 2008). We now report an extensive mutational analysis which identifies four variable residues (294, 296, 396, and 404) as the main determinants of the different syncytial phenotypes found among hMPV F proteins. These residues lie near two conserved histidines (H368 and H435) in a three-dimensional (3D) model of the pretriggered hMPV F trimer. Mutagenesis of H368 and H435 indicates that protonation of these histidines (particularly His435) is a key event to destabilize the hMPV F proteins that require low pH for cell-cell fusion. The syncytial phenotypes were reproduced in cells infected with the corresponding hMPV strains. However, the low-pH dependency for syncytium formation could not be related with a virus entry pathway dependent on an acidic environment. It is postulated that low pH may be acting for some hMPV strains as certain destabilizing mutations found in unusual strains of other paramyxoviruses. In any case, the results presented here and those reported by Schowalter et al. (J. Virol. 83:1511-1522, 2009) highlight the relevance of certain residues in the linker region and domain II of the pretriggered hMPV F protein for the process of membrane fusion.     

Foamy virus envelope glycoprotein-mediated entry involves a pH-dependent fusion process

In general, enveloped viruses use two different entry strategies and are classified accordingly into pH-dependent and pH-independent viruses. Different members of the retrovirus family use one or the other strategy. Little is known about the uptake of foamy viruses (FV), a special group of retroviruses, into the target cells. In this study, we examined the pH dependence of FV entry by analyzing FV envelope glycoprotein (Env)-mediated infection of target cells with murine leukemia virus or FV vector pseudotypes in the presence of various lysosomotropic agents. Similar to vesicular stomatitis virus glycoprotein G (VSV-G)-mediated uptake, FV Env-mediated entry was inhibited by various lysosomotropic agents, suggesting a pH-dependent endocytic pathway. However, in contrast to its effect on VSV-G pseudotypes, chloroquine failed to reduce the infectivity of FV Env pseudotypes, implying that the pathway is different from that of VSV-G. Glycoproteins of various other FV species showed inhibition profiles similar to that of the prototype FV (PFV) Env. Analysis of the pH dependence of the FV Env-mediated fusion process in a cell-to-cell fusion assay revealed an induction of syncytium formation by a short exposure to acidic pH, peaking around pH 5.5. Interestingly, of all FV Env species analyzed, only the PFV Env had a significant fusion activity at neutral pH. Taken together, these data suggest a pH-dependent endocytic pathway for infection of target cells by FV.     

Regulation of human immunodeficiency virus type 1 envelope glycoprotein fusion by a membrane-interactive domain in the gp41 cytoplasmic tail

Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.     

Role of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein in cell-cell fusion and virus infection

The membrane-spanning domain (MSD) of the human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein is critical for its biological activity. Previous C-terminal truncation studies have predicted an almost invariant core structure of 12 amino acid residues flanked by basic amino acids in the HIV-1 MSD that function to anchor the glycoprotein in the lipid bilayer. To further understand the role of specific amino acids within the MSD core, we initially replaced the core region with 12 leucine residues and then constructed recovery-of-function mutants in which specific amino acid residues (including a GGXXG motif) were reintroduced. We show here that conservation of the MSD core sequence is not required for normal expression, processing, intracellular transport, and incorporation into virions of the envelope glycoprotein (Env). However, the amino acid composition of the MSD core does influence the ability of Env to mediate cell-cell fusion and plays a critical role in the infectivity of HIV-1. Replacement of conserved amino acid residues with leucine blocked virus-to-cell fusion and subsequent viral entry into target cells. This restriction could not be released by C-terminal truncation of the gp41 glycoprotein. These studies imply that the highly conserved core residues of the HIV Env MSD, in addition to serving as a membrane anchor, play an important role in mediating membrane fusion during viral entry.     

Transformation of rodent fibroblasts by the jaagsiekte sheep retrovirus envelope is receptor independent and does not require the surface domain

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). Expression of the JSRV envelope protein (Env) is sufficient to transform immortalized and primary fibroblasts, but the precise mechanisms of this process are not known. The cellular receptor for JSRV is hyaluronidase 2 (Hyal-2), the product of a putative tumor suppressor gene that in humans maps to a chromosomal region frequently deleted in the development of lung and breast cancers. Here we report studies to determine whether the Hyal-2-JSRV Env interaction plays a role in virus-induced transformation of rodent fibroblasts. Chimeric Env proteins between JSRV and the unrelated murine retroviruses Moloney murine leukemia virus (MMuLV) and mouse mammary tumor virus (MMTV) showed cell surface expression comparable to that of wild-type MMuLV Env and rescued infection of MMuLV particle pseudotypes. Interestingly, an MMuLV-JSRV chimera in which the putative receptor binding domain (RBD) and proline-rich region (PRR) of JSRV Env were replaced by the RBD and PRR of MMuLV induced transformation of 208F, a rodent fibroblast line. Cell lines derived from foci of MMuLV-JSRV chimera-transformed 208F cells grew in soft agar and showed Akt activation, a hallmark of JSRV-transformed rodent fibroblasts. Transformation assays performed using proteins with amino-terminal deletion mutations showed that the carboxy-terminal 141 amino acids of the transmembrane subunit (TM) were sufficient to induce cell transformation when targeted to the membrane with a myristoylation signal. Thus, the JSRV TM is necessary and sufficient to transform rodent fibroblasts. Taken together these results indicate that the interaction with Hyal-2 at least is not an essential determinant of JSRV-induced transformation of fibroblasts and that the viral TM functions essentially as an oncoprotein.     

Mutations in the cytoplasmic domain of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion

SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.     

Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion.

The baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus (BV) and is involved in BV entry into the host cell by endocytosis. To determine whether gp64 alone was sufficient to mediate membrane fusion, the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 protein was transiently expressed in uninfected insect cells. Cells expressing the baculovirus gp64 protein were examined for membrane fusion activity by using a syncytium formation assay under various conditions of exposure to low pH. Cells expressing the gp64 protein mediated membrane fusion and syncytium formation in a pH-dependent manner. A pH of 5.5 or lower was required to induce membrane fusion. In addition, exposure of gp64-expressing cells to low pH for as little as 5 s was sufficient to induce gp64-mediated syncytium formation. These studies provide direct evidence that gp64 is a pH-dependent membrane fusion protein and suggest that gp64 is the protein responsible for fusion of the virion envelope with the endosome membrane during BV entry into the host cell by endocytosis.     

Mutations in multiple domains activate paramyxovirus F protein-induced fusion

SER virus, a paramyxovirus that is closely related to simian virus 5 (SV5), is unusual in that it fails to induce syncytium formation. The SER virus F protein has an unusually long cytoplasmic tail (CT), and it was previously observed that truncations or specific mutations of this domain result in enhanced syncytium formation. In addition to the long CT, the SER F protein has nine amino acid differences from the F protein of SV5. We previously observed only a partial suppression of fusion in a chimeric SV5 F protein with a CT derived from SER virus, indicating that these other amino acid differences between the SER and SV5 F proteins also play a role in regulating the fusion phenotype. To examine the effects of individual amino acid differences, we mutated the nine SER residues individually to the respective residues of the SV5 F protein. We found that most of the mutants were expressed well and were transported to the cell surface at levels comparable to that of the wild-type SER F protein. Many of the mutants showed enhanced lipid mixing, calcein transfer, and syncytium formation even in the presence of the long SER F protein CT. Some mutants, such as the I310 M, T438S, M489I, T516V, and N529K mutants, also showed fusion at lower temperatures of 32, 25, and 18 degrees C. The residue Asn529 plays a critical role in the suppression of fusion activity, as the mutation of this residue to lysine caused a marked enhancement of fusion. The effect of the N529K mutation on the enhancement of fusion by a previously described mutant, L539,548A, as well as by chimeric SV5/SER F proteins was also dramatic. These results indicate that activation to a fusogenic conformation is dependent on the interplay of residues in the ectodomain, the transmembrane domain, and the CT domain of paramyxovirus F proteins.     

Low-pH activation of Bacillus cereus spores

Heat activation of bacterial spores at low pH was investigated in detail. Unlike activation of spores in distilled water at a neutral pH, activation at low pH involves two superimposed processes: enhanced activation and death. Low-pH-activated spores failed to germinate in d-alanine, in contrast to spores activated at neutral pH, owing to the abolition of alanine-racemase activity. Morphological and permeability changes such as release and partial disruption of spores were dipicolinic acid-observed during low-pH activation. The kinetics pattern of low-pH activation, as well as the change in properties of the spores thereafter, suggest that the mechanism of low-pH activation differs from that of other kinds of heat-activation.