Rebecca Ellis Dutch

Structural Basis for Paramyxovirus-Mediated Membrane Fusion

Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers.

Cathepsin L Is Involved in Proteolytic Processing of the Hendra Virus Fusion Protein

ABSTRACT Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F0) to the active F1 + F2 disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F0 into F1 and F2 fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.

Viral entry mechanisms: the increasing diversity of paramyxovirus entry

The paramyxovirus family contains established human pathogens such as the measles virus and human respiratory syncytial virus, as well as emerging pathogens including the Hendra and Nipah viruses and the recently identified human metapneumovirus. Two major envelope glycoproteins, the attachment protein and the fusion protein, promote the processes of viral attachment and virus‐cell membrane fusion required for entry. Although common mechanisms of fusion protein proteolytic activation and the mechanism of membrane fusion promotion have been shown in recent years, considerable diversity exists in the family relating to receptor binding and the potential mechanisms of fusion triggering.

Virus membrane fusion proteins: biological machines that undergo a metamorphosis.

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structuralelements in the processing and function of these viral fusion proteins is discussed.

Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH

ABSTRACT Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.

Paramyxovirus Fusion and Entry: Multiple Paths to a Common End

The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, the parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the zoonotic henipaviruses, Hendra and Nipah. While the expression of a type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses, there is considerable variation in viral attachment, the activation and triggering of the fusion protein, and the process of viral entry. In this review, we discuss recent advances in the understanding of paramyxovirus F protein-mediated membrane fusion, an essential process in viral infectivity. We also review the role of the other surface glycoproteins in receptor binding and viral entry, and the implications for viral infection. Throughout, we concentrate on the commonalities and differences in fusion triggering and viral entry among the members of the family. Finally, we highlight key unanswered questions and how further studies can identify novel targets for the development of therapeutic treatments against these human pathogens.

LOW PH TRIGGERING OF HUMAN METAPNEUMOVIRUS FUSION: ESSENTIAL RESIDUES AND IMPORTANCE IN ENTRY

ABSTRACT Human metapneumovirus (HMPV) is a significant respiratory pathogen classified in the Pneumovirinae subfamily of the paramyxovirus family. Recently, we demonstrated that HMPV F protein-promoted cell-cell fusion is stimulated by exposure to low pH, in contrast to what is observed for other paramyxovirus F proteins. In the present study, we examined the potential role of histidine protonation in HMPV F fusion and investigated the role of low pH in HMPV viral entry. Mutagenesis of the three ectodomain histidine residues of the HMPV F protein demonstrated that the mutation of a histidine in the heptad repeat B linker domain (H435) ablated fusion activity without altering cell surface expression or proteolytic processing significantly. Modeling of the HMPV F protein revealed several basic residues surrounding this histidine residue, and the mutation of these residues also reduced fusion activity. These results suggest that electrostatic repulsion in the heptad repeat B linker region may contribute to the triggering of HMPV F. In addition, we examined the effect of inhibitors of endosomal acidification or endocytosis on the entry of a recombinant green fluorescent protein-expressing HMPV. Interestingly, chemicals that raise the pH of endocytic vesicles resulted in a 30 to 50% decrease in HMPV infection, while the inhibitors of endocytosis reduced infection by as much as 90%. These data suggest that HMPV utilizes an endocytic entry mechanism, in contrast to what has been hypothesized for most paramyxoviruses. In addition, our results indicate that HMPV uses the low pH of the endocytic pathway to enhance infectivity, though the role of low pH likely differs from classically described mechanisms.

Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

ABSTRACT The Hendra virus fusion (F) protein is synthesized as a precursor protein, F0, which is proteolytically processed to the mature form, F1+F2. Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca2+, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXΦ. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.

Human Metapneumovirus (HMPV) Binding and Infection Are Mediated by Interactions between the HMPV Fusion Protein and Heparan Sulfate

ABSTRACT Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), specifically, heparan sulfate proteoglycans (HSPGs), were resistant to binding and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and small hydrophobic (SH) proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding to cells did not require β1 integrin expression, and RGD-mediated interactions were not essential in promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking β1 integrin, however, were less permissive to HMPV infection, indicating that while β1 integrins play an important role in promoting HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F to heparan sulfate proteoglycans.

Deletion of the Cytoplasmic Tail of the Fusion Protein of the Paramyxovirus Simian Virus 5 Affects Fusion Pore Enlargement

ABSTRACT The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail− or FΔ19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail− mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail− mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail− is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.