Corrigendum to Effects of retroviral envelope-protein cleavage upon trafficking, incorporation, and membrane fusion, Virology 405 (2010) Pages 214–224

ABSTRACT Mumps virus (MuV), an enveloped RNA virus of the Paramyxoviridae family and the causative agent of mumps, affects the salivary glands and other glandular tissues as well as the central nervous system. The virus enters the cell by inducing the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by MuV envelope proteins: the hemagglutinin-neuraminidase and fusion (F) protein. Cleavage of the MuV F protein (MuV-F) into two subunits by the cellular protease furin is a prerequisite for fusion and virus infectivity. Here, we show that 293T (a derivative of HEK293) cells do not produce syncytia upon expression of MuV envelope proteins or MuV infection. This failure is caused by the inefficient MuV-F cleavage despite the presence of functional furin in 293T cells. An expression cloning strategy revealed that overexpression of lysosome-associated membrane proteins (LAMPs) confers on 293T cells the ability to produce syncytia upon expression of MuV envelope proteins. The LAMP family comprises the ubiquitously expressed LAMP1 and LAMP2, the interferon-stimulated gene product LAMP3, and the cell type-specific proteins. The expression level of the LAMP3 gene, but not of LAMP1 and LAMP2 genes, differed markedly between 293T and HEK293 cells. Overexpression of LAMP1, LAMP2, or LAMP3 allowed 293T cells to process MuV-F efficiently. Furthermore, these LAMPs were found to interact with both MuV-F and furin. Our results indicate that LAMPs support the furin-mediated cleavage of MuV-F and that, among them, LAMP3 may be critical for the process, at least in certain cells. IMPORTANCE The cellular protease furin mediates proteolytic cleavage of many host and pathogen proteins and plays an important role in viral envelope glycoprotein maturation. MuV, an enveloped RNA virus of the Paramyxoviridae family and an important human pathogen, enters the cell through the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by the viral attachment protein and the F protein. Cleavage of MuV-F into two subunits by furin is a prerequisite for fusion and virus infectivity. Here, we show that LAMPs support the furin-mediated cleavage of MuV-F. Expression levels of LAMPs affect the processing of MuV-F and MuV-mediated membrane fusion. Among LAMPs, the interferon-stimulated gene product LAMP3 is most critical in certain cells. Our study provides potential targets for anti-MuV therapeutics.

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Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64

Journal of Virology ◽

10.1128/jvi.76.11.5729-5736.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5729-5736 ◽

Cited By ~ 89

Author(s):

Oliver Lung ◽

Marcel Westenberg ◽

Just M. Vlak ◽

Douwe Zuidema ◽

Gary W. Blissard

Keyword(s):

Membrane Fusion ◽

Envelope Protein ◽

Protein Gene ◽

Autographa Californica ◽

Viral Envelope ◽

Viral Attachment ◽

F Protein ◽

Viral Propagation ◽

The Family ◽

Vesicular Stomatitis

ABSTRACT GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.

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The first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.88.9.3623 ◽

1991 ◽

Vol 88 (9) ◽

pp. 3623-3627 ◽

Cited By ~ 70

Author(s):

A. E. Spruce ◽

A. Iwata ◽

W. Almers

Keyword(s):

Membrane Fusion ◽

Envelope Protein ◽

Viral Envelope ◽

Viral Envelope Protein

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Structure-Based Mutational Analysis of Several Sites in the E Protein: Implications for Understanding the Entry Mechanism of Japanese Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.00293-15 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5668-5686 ◽

Cited By ~ 22

Author(s):

Haibin Liu ◽

Yi Liu ◽

Shaobo Wang ◽

Yanjun Zhang ◽

Xiangyang Zu ◽

...

Keyword(s):

Amino Acids ◽

Japanese Encephalitis Virus ◽

Membrane Fusion ◽

Japanese Encephalitis ◽

Receptor Binding ◽

Viral Entry ◽

Envelope Protein ◽

Encephalitis Virus ◽

Entry Pathway ◽

Entry Mechanism

ABSTRACTJapanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations—in I0, ij, BC, and FG and the R9A substitution—impaired virus assembly, and the assembled virus particles of another five mutations—in kl and the E373A, F407A, L221S, and W217A substitutions—were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity.IMPORTANCERecently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses.

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Structure of the dengue virus envelope protein after membrane fusion

Nature ◽

10.1038/nature02165 ◽

2004 ◽

Vol 427 (6972) ◽

pp. 313-319 ◽

Cited By ~ 759

Author(s):

Yorgo Modis ◽

Steven Ogata ◽

David Clements ◽

Stephen C. Harrison

Keyword(s):

Dengue Virus ◽

Membrane Fusion ◽

Envelope Protein ◽

Virus Envelope ◽

Virus Envelope Protein

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A Dissection of Steps Leading to Viral Envelope Protein-Mediated Membrane Fusion

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1991.tb36499.x ◽

1991 ◽

Vol 635 (1 Calcium Entry) ◽

pp. 285-296 ◽

Cited By ~ 29

Author(s):

ROBERT BLUMENTHAL ◽

CHRISTIAN SCHOCH ◽

ANU PURI ◽

MICHAEL J. CLAGUE

Keyword(s):

Membrane Fusion ◽

Envelope Protein ◽

Viral Envelope ◽

Viral Envelope Protein

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TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus

Journal of Virology ◽

10.1128/jvi.01567-16 ◽

2016 ◽

Vol 90 (24) ◽

pp. 11231-11246 ◽

Cited By ~ 9

Author(s):

Bingling Yun ◽

Yao Zhang ◽

Yongzhen Liu ◽

Xiaolu Guan ◽

Yongqiang Wang ◽

...

Keyword(s):

Viral Replication ◽

Membrane Fusion ◽

Serine Protease ◽

Viral Envelope ◽

Host Cells ◽

Protein Cleavage ◽

Avian Metapneumovirus ◽

F Protein ◽

Subtype B ◽

Transmembrane Serine Protease

ABSTRACTThe entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV.IMPORTANCEProteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases usedin vitroandvivoare not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

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Role of the Ectodomain of the gp41 Transmembrane Envelope Protein of Human Immunodeficiency Virus Type 1 in Late Steps of the Membrane Fusion Process

Journal of Virology ◽

10.1128/jvi.78.2.811-820.2004 ◽

2004 ◽

Vol 78 (2) ◽

pp. 811-820 ◽

Cited By ~ 47

Author(s):

Séverine Bär ◽

Marc Alizon

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Envelope Protein ◽

Target Cells ◽

Loop Region ◽

Immunodeficiency Virus ◽

Fluorescent Cells ◽

Hiv 1

ABSTRACT The membrane fusion process mediated by the gp41 transmembrane envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) was addressed by a flow cytometry assay detecting exchanges of fluorescent membrane probes (DiI and DiO) between cells expressing the HIV-1 envelope proteins (Env) and target cells. Double-fluorescent cells were detected when target cells expressed the type of chemokine receptor, CXCR4 or CCR5, matching the type of gp120 surface envelope protein, X4 or R5, respectively. Background levels of double-fluorescent cells were observed when the gp120-receptor interaction was blocked by AMD3100, a CXCR4 antagonist. The L568A mutation in the N-terminal heptad repeat (HR1) of gp41 resulted in parallel inhibition of the formation of syncytia and double-fluorescent cells, indicating that gp41 had a direct role in the exchange of fluorescent probes. In contrast, three mutations in the loop region of the gp41 ectodomain, located on either side of the Cys-(X)5-Cys motif (W596 M and W610A) or at the distal end of HR1 (D589L), had limited or no apparent effect on membrane lipid mixing between Env+ and target cells, while they blocked formation of syncytia and markedly reduced the exchanges of cytoplasmic fluorescent probes. The loop region could therefore have a direct or indirect role in events occurring after the merging of membranes, such as the formation or dilation of fusion pores. Two types of inhibitors of HIV-1 entry, the gp41-derived peptide T20 and the betulinic acid derivative RPR103611, had limited effects on membrane exchanges at concentrations blocking or markedly reducing syncytium formation. This finding confirmed that T20 can inhibit the late steps of membrane fusion (post-lipid mixing) and brought forth an indirect argument for the role of the gp41 loop region in these steps, as mutations conferring resistance to RPR103611V were mapped in this region (I595S or L602H).

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