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.

The emergence of a new coronaviral respiratory disease calls for repurposing existing drugs

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.

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 (F 0 ) to the active F 1 ؉ F 2 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 pCAGGSHendra 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 F 0 into F 1 and F 2 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.

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.