Tryptophan-based motifs in the LLP3 region of the HIV-1 envelope glycoprotein cytoplasmic tail direct trafficking to the endosomal recycling compartment and mediate particle incorporation.

IMPORTANCE: The HIV-1 envelope glycoprotein (Env) is an essential component of the virus and has an exceedingly long cytoplasmic tail (CT). Previous studies have suggested that trafficking signals in the CT interact with host factors to regulate the incorporation of Env into particles. One particular area of interest is termed lentiviral lytic peptide 3 (LLP3), as small deletions in this region have been shown to disrupt Env incorporation. In this study, we identify a small region within LLP3 that regulates how Env associates with cellular recycling compartments. Mutants that reduced or eliminated Env from the recycling compartment also reduced Env incorporation into particles. These findings emphasize the importance of two tryptophan motifs in LLP3 for the incorporation of Env into particles and provide additional support for the idea that the CT interacts with host recycling pathways to determine particle incorporation.

Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Rab11-FIP1C Is Dispensable for HIV-1 Replication in Primary CD4+ T Cells, but Its Role Is Cell Type Dependent in Immortalized Human T-Cell Lines.

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4+ T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.

Mutagenesis of the di-leucine motif in the cytoplasmic tail of newcastle disease virus fusion protein modulates the viral fusion ability and pathogenesis.

BACKGROUND: Newcastle disease virus (NDV) is a highly infectious viral disease, which can affect chickens and many other kinds of birds. The main virulence factor of NDV, the fusion (F) protein, is located on the viral envelope and plays a major role in the virus' ability to penetrate cells and cause host cell fusion during infection. Multiple highly conserved tyrosine and di-leucine (LL) motifs in the cytoplasmic tail (CT) of the virus may contribute to F protein functionality in the viral life cycle. METHODS: To examine the contribution of the LL motif in the biosynthesis, transport, and function of the F protein, we constructed and rescued a NDV mutant strain, rSG10*-F/L537A, with an L537A mutation using a reverse genetic system. Subsequently, we compared the differences in the syncytium formation ability, pathogenicity, and replication levels of wild-type rSG10* and the mutated strain. RESULTS: Compared with rSG10*, rSG10*-F/L537A had attenuated syncytial formation and pathogenicity, caused by a viral budding defect. Further studies showed that the LL-motif mutation did not affect the replication, transcription, or translation of the virus genome but affected the expression of the F protein at the cell surface. CONCLUSIONS: We concluded that the LL motif in the NDV F CT affected the regulation of F protein expression at the cell surface, thus modulating the viral fusion ability and pathogenic phenotype.

A basic charge cluster near the C-terminus of the cytoplasmic tail contributes to the molecular stability of human herpesvirus 8 E3 ubiquitin ligases.

Human herpesvirus 8 (HHV8; also known as Kaposi's sarcoma-associated herpesvirus [KSHV]) utilizes the viral E3 ubiquitin ligase family members K3 and K5 for immune evasion. Both K3 and K5 mediate the ubiquitination of host MHC class I (MHC-I) molecules, which play a key role in antigen presentation to cytotoxic T lymphocytes (CTLs). Because ubiquitinated MHC-I is immediately down-regulated from the cell surface, HHV8-infected cells can escape surveillance by CTLs. K3 and K5 have similar domain structures and topologies. They contain an N-terminal RINGv ubiquitin ligase domain, two transmembrane helices, and an intrinsically disordered cytoplasmic tail at the C-terminus. The cytoplasmic tail contains a membrane-proximal "conserved region" involved in ligase activity. On the other hand, the role of the membrane-distal region of the cytoplasmic tail, termed the "C-tail" in this study, remains unclear. Here, we demonstrate that the C-tail contributes to the protein expression of both K3 and K5. The C-tail-truncated K3 and K5 mutants were rapidly reduced in cells. The recombinant C-tail proteins bind to acidic lipids via a basic charge cluster located near the C-terminus of the C-tails. Similar to the C-tail-truncated mutants, the basic charge cluster-substituting mutants showed decreased protein expression of K3 and K5. These findings suggest that the basic charge cluster near the C-terminus of the cytoplasmic tail contributes to the molecular stability of K3 and K5.

Structural characterization of HIV-1 matrix mutants implicated in envelope incorporation.

During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation. Conversely, Env incorporation can be rescued by a compensatory mutation in the MA trimer interface (Q63R). The impact of these MA mutations on the structure and trimerization properties of MA is not known. In this study, we employed NMR spectroscopy, X-ray crystallography, and sedimentation techniques to characterize the structure and trimerization properties of HIV-1 MA A45E, Q63R, T70R, and L75G mutant proteins. NMR data revealed that these point mutations did not alter the overall structure and folding of MA but caused minor structural perturbations in the trimer interface. Analytical ultracentrifugation data indicated that mutations had a minimal effect on the MA monomer-trimer equilibrium. The high-resolution X-ray structure of the unmyristoylated MA Q63R protein revealed hydrogen bonding between the side chains of adjacent Arg-63 and Ser-67 on neighboring MA molecules, providing the first structural evidence for an additional intermolecular interaction in the trimer interface. These findings advance our knowledge of the interplay of MA trimerization and Env incorporation into HIV-1 particles.

Truncation of the Cytoplasmic Tail of Equine Infectious Anemia Virus Increases Virion Production by Improving Env Cleavage and Plasma Membrane Localization.

Envelope glycoproteins (Envs) of lentiviruses harbor unusually long cytoplasmic tails (CTs). Natural CT truncations always occur in vitro and are accompanied by attenuated virulence, but their effects on viral replication have not been fully elucidated. The Env in equine infectious anemia virus (EIAV) harbors the longest CT in the lentiviral family, and a truncated CT was observed in a live attenuated vaccine. This study demonstrates that CT truncation significantly increased EIAV production, as determined by comparing the virion yields from EIAV infectious clones in the presence and absence of the CT. A significant increase in a cleaved product from the CT-truncated Env precursor, but not the full-length Env, was observed. We further confirmed that the presence of the CT inhibited the cleavage of the Env precursor and found that a functional domain located at the C terminus was responsible for this function. Moreover, CT-truncated Env was mainly localized at the plasma membrane (PM), while full-length Env was mainly localized in the cytoplasm. The CT truncation caused a dramatic reduction in the endocytosis of Env. These results suggest that the CT can modulate the processing and trafficking of EIAV Env and thus regulate EIAV replication. IMPORTANCE The mature lentivirus envelope glycoprotein (Env) is composed of a surface unit (SU) and a transmembrane unit (TM), which are cleaved products of the Env precursor. After mature Env is heterodimerically formed from the cleavage of the Env precursor, it is trafficked to the plasma membrane (PM) for incorporation and virion assembly. Env harbors a long cytoplasmic tail (CT), which has been increasingly found to play multiple roles in the Env biological cycle. Here, we revealed for the first time that the CT of equine infectious anemia virus (EIAV) Env inhibits cleavage of the Env precursor. Simultaneously, the CT promoted Env endocytosis, resulting in weakened Env localization at the PM. We also validated that the CT could significantly decrease EIAV production. These findings suggest that the CT regulates the processing and trafficking of EIAV Env to balance virion production.

Cytoplasmic Tail Truncation of SARS-CoV-2 Spike Protein Enhances Titer of Pseudotyped Vectors but Masks the Effect of the D614G Mutation.

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein's function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.

The cytoplasmic tail substitution increases the assembly efficiency of Ebola virus glycoprotein on the budded virus of Bombyx mori nucleopolyhedrovirus.

Glycoprotein (GP1,2) of the Ebola virus (EBOV) is the key membrane fusion protein, which is a key candidate protein for vaccine preparations. Previously, GP1,2 was expressed by Bombyx mori nucleopolyhedrovirus (BmNPV) expression vector system; however, few GP1,2 was incorporated into budded virus (BV) of BmNPV. To improve the incorporation efficiency of GP1,2 into the virion, the GP1,2 fusion with the cytoplasmic tail of GP64 of BmNPV was expressed in BmN cells by the BmNPV expression system. The BV was purified by ultracentrifugation, and GP1,2 expression in BV was detected by the antibody. The result indicated that a 532% increase in the relative GP1,2 densitometry signal was observed in constructs utilizing the GP64 C-terminal domain; moreover, the substitution of GP1,2 native signal peptide with GP64 signal peptide increased the incorporation efficiency by 34.6% in the relative GP1,2 densitometry signal. We revealed that the application of the cytoplasmic tail of BmNPV GP64 significantly increased the incorporation rate of GP1,2 into the BV envelope. This study lays a foundation for GP1,2 vaccine development.

Interaction Interface of Mason-Pfizer Monkey Virus Matrix and Envelope Proteins.

Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

Elucidating the Basis for Permissivity of the MT-4 T-Cell Line to Replication of an HIV-1 Mutant Lacking the gp41 Cytoplasmic Tail.

HIV-1 encodes an envelope glycoprotein (Env) that contains a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virus particles and viral transmission. In most physiologically relevant cell types, the gp41 CT is required for HIV-1 replication, but in the MT-4 T-cell line the gp41 CT is not required for a spreading infection. To help elucidate the role of the gp41 CT in HIV-1 transmission, in this study, we investigated the viral and cellular factors that contribute to the permissivity of MT-4 cells to gp41 CT truncation. We found that the kinetics of HIV-1 production and virus release are faster in MT-4 than in the other T-cell lines tested, but MT-4 cells express equivalent amounts of HIV-1 proteins on a per-cell basis relative to cells not permissive to CT truncation. MT-4 cells express higher levels of plasma-membrane-associated Env than nonpermissive cells, and Env internalization from the plasma membrane is less efficient than that from another T-cell line, SupT1. Paradoxically, despite the high levels of Env on the surface of MT-4 cells, 2-fold less Env is incorporated into virus particles produced from MT-4 than SupT1 cells. Contact-dependent transmission between cocultured 293T and MT-4 cells is higher than in cocultures of 293T with most other T-cell lines tested, indicating that MT-4 cells are highly susceptible to cell-to-cell infection. These data help to clarify the long-standing question of how MT-4 cells overcome the requirement for the HIV-1 gp41 CT and support a role for gp41 CT-dependent trafficking in Env incorporation and cell-to-cell transmission in physiologically relevant cell lines.IMPORTANCE The HIV-1 Env cytoplasmic tail (CT) is required for efficient Env incorporation into nascent particles and viral transmission in primary CD4+ T cells. The MT-4 T-cell line has been reported to support multiple rounds of infection of HIV-1 encoding a gp41 CT truncation. Uncovering the underlying mechanism of MT-4 T-cell line permissivity to gp41 CT truncation would provide key insights into the role of the gp41 CT in HIV-1 transmission. This study reveals that multiple factors contribute to the unique ability of a gp41 CT truncation mutant to spread in cultures of MT-4 cells. The lack of a requirement for the gp41 CT in MT-4 cells is associated with the combined effects of rapid HIV-1 protein production, high levels of cell-surface Env expression, and increased susceptibility to cell-to-cell transmission compared to nonpermissive cells.

A common CORIN variant in hypertension reduces corin intracellular trafficking by exposing an inhibitory N-terminus.

Corin is a transmembrane serine protease that activates atrial natriuretic peptide, a cardiac hormone essential for normal blood pressure. Corin is synthesized as a zymogen and activated on the cell surface. In previous studies, we identified a CORIN variant allele with an adenine insertion in the 5'-end of the coding region in ∼5% of hypertensive individuals in a Chinese population. The protein, named insA, encoded by the CORIN variant allele has a shortened cytoplasmic tail and reduced atrial natriuretic peptide processing activity. It remains unknown how a shortened cytoplasmic tail impairs corin function. In this study, we expressed a series of corin mutants with different N-terminal sequences and analyzed them by Western blotting, flow cytometry, protein chase, and immunostaining. Our results revealed that a Gly-Asn sequence after the initiating Met at the newly generated N-terminus was responsible for delaying corin trafficking in the Golgi. Deletion of the N-terminal Gly and Asn residues increased the intracellular trafficking, cell surface expression, and activation cleavage of the insA variant. These results help to explain how the CORIN variant allele impairs corin structure and function as an underlying mechanism in hypertension.

Sequence Determinants in Gammaretroviral Env Cytoplasmic Tails Dictate Virus-Specific Pseudotyping Compatibility.

Viruses can incorporate foreign glycoproteins to form infectious particles through a process known as pseudotyping. However, not all glycoproteins are compatible with all viruses. Despite the fact that viral pseudotyping is widely used, what makes a virus/glycoprotein pair compatible is poorly understood. To study this, we chose to analyze a gammaretroviral glycoprotein (Env) whose compatibility with different viruses could be modulated through small changes in its cytoplasmic tail (CT). One form of this glycoprotein is compatible with murine leukemia virus (MLV) particles but incompatible with human immunodeficiency virus type 1 (HIV-1) particles, while the second is compatible with HIV-1 particles but not with MLV particles. To decipher the factors affecting virus-specific Env incompatibility, we characterized Env incorporation, maturation, cell-to-cell fusogenicity, and virus-to-cell fusogenicity of each Env. The HIV-1 particle incompatibility correlated with less efficient cleavage of the R peptide by HIV-1 protease. However, the MLV particle incompatibility was more nuanced. MLV incompatibility appeared to be caused by lack of incorporation into particles, yet incorporation could be restored by further truncating the CT or by using a chimeric MLV Gag protein containing the HIV-1 MA without fully restoring infectivity. The MLV particle incompatibility appeared to be caused in part by fusogenic repression in MLV particles through an unknown mechanism. This study demonstrates that the Env CT can dictate functionality of Env within particles in a virus-specific manner.IMPORTANCE Viruses utilize viral glycoproteins to efficiently enter target cells during infection. How viruses acquire viral glycoproteins has been studied to understand the pathogenesis of viruses and develop safer and more efficient viral vectors for gene therapies. The CTs of viral glycoproteins have been shown to regulate various stages of glycoprotein biogenesis, but a gap still remains in understanding the molecular mechanism of glycoprotein acquisition and functionality regarding the CT. Here, we studied the mechanism of how specific mutations in the CT of a gammaretroviral envelope glycoprotein distinctly affect infectivity of two different viruses. Different mutations caused failure of glycoproteins to function in a virus-specific manner due to distinct fusion defects, suggesting that there are virus-specific characteristics affecting glycoprotein functionality.

Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers.

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.IMPORTANCE The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.

Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress.

Nipah virus (NiV), a paramyxovirus in the genus Henipavirus, has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F) and, to a much lesser extent, the attachment glycoprotein (G) autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXØ or dityrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery.IMPORTANCE Henipaviruses can cause deadly infections of medical, veterinary, and agricultural importance. With recent discoveries of new henipa-like viruses, understanding the mechanisms by which these viruses reproduce is paramount. We have focused this study on identifying the functional interactions of three Nipah virus proteins during viral assembly and particularly on the role of one of these proteins, the fusion glycoprotein, in the incorporation of other viral proteins into viral particles. By identifying several regions in the fusion glycoprotein that drive viral assembly, we further our understanding of how these viruses assemble and egress from infected cells. The results presented will likely be useful toward designing treatments targeting this aspect of the viral life cycle and for the production of new viral particle-based vaccines.

Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16.

MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium.

The IgG1 B-cell receptor provides survival and proliferative signals analogue to the Igα but not the Igß co-receptor.

The function of the IgM B-cell receptor (BCR) is dependent on intact signaling of the co-receptors Igα and Igß, both of which contain a cytoplasmic tail bearing an immunoreceptor tyrosine-based activation motif. We have previously demonstrated that the cytoplasmic tail of the IgG1 BCR can partially compensate for the loss of the signaling moiety of Igα. Here, we show that unlike Igα, Igß signaling is indispensable for the development and function of IgG1-expressing B cells. Deletion of the cytoplasmic signaling tail of Igß compromised the survival and proliferation not only of IgM(+) B cells but also of IgG1-expressing B cells. In the absence of the signaling tail of Igß, the transcription levels of the antiapoptotic gene bcl-xl and the cell-cycle gene ccnd2 were reduced, consistent with the observed defects in survival and proliferation. These results demonstrate functional differences between Igα and Igß in the transduction of IgG1 BCR signal.

[VLP vaccines and effects of HIV-1 Env protein modifications on their antigenic properties].

An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV.

A novel cytoplasmic tail motif regulates mouse corin expression on the cell surface.

Type II transmembrane serine proteases (TTSPs) are important in many biological processes. Cell surface expression is critical for TTSP activation and function. To date, the mechanism underlying TTSP cell surface expression is poorly understood. Corin is a TTSP and acts as the pro-atrial natriuretic peptide convertase that is essential for sodium homeostasis and normal blood pressure. In this study, we investigated how cytoplasmic tail sequences may regulate corin expression and activation on the cell surface. By site-directed mutagenesis, we made mouse corin proteins with truncations or point-mutations in the cytoplasmic tail. We expressed the mutants in transfected HEK293 cells and analyzed corin cell surface expression and activation by Western blotting and flow cytometry. We found that corin truncation mutants lacking a Lys-Phe-Gln sequence at residues 71-73 had higher levels of cell surface expression and activation compared with that in wild-type corin. When Lys-71, Phe-72 and Gln-73 residues were mutated together, but not individually, in corin with the full-length cytoplasmic tail, increased levels of cell surface expression and zymogen activation were also observed. These results indicate that residues Lys-71, Phe-72 and Gln-73 serve as a novel retention motif in the intracellular pathway to regulate corin cell surface expression and activation.

An amphipathic sequence in the cytoplasmic tail of HIV-1 Env alters cell tropism and modulates viral receptor specificity.

The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from a CD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying a truncated Env (CT84). To determine whether a specific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared a series of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha- helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred a switch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.