Virus Activation by Host Proteinases. A Pivotal Role in the Spread of Infection, Tissue Tropism and Pathogenicity

Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible Ͼ2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions.IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane.KEYWORDS conformational changes, glycoprotein B, herpes simplex viruses, herpesviruses, inactivation, low pH, membrane fusion

Section: Discussionmentioning

confidence: 99%

Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry

Weed

Pritchard

Gonzalez

et al. 2017

“…Besides the restricted expression of cellular receptors, limited expression of activating proteases is an important factor for virus spread and cell tropism, thereby determining pathogenesis in vivo (8,33,38,49,54). Our results showing that NiV can use two broadly expressed cathepsins, however, suggest that protease expression does not restrict NiV spread in vivo.…”

Section: Discussionmentioning

confidence: 99%

Activation of the Nipah Virus Fusion Protein in MDCK Cells Is Mediated by Cathepsin B within the Endosome-Recycling Compartment

Diederich

Sauerhering

Weis

et al. 2012

c Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV.

“…Thus, viruses with multibasic amino acids at the cleavage site are considered virulent, and those characterized by monobasic amino acids at the cleavage site are avirulent (3,4,5,6). Cleavage of F0 polypeptides into F2 and F1 is essential for a virus particle to become infectious (31). In chickens, the F protein of pathogenic NDV is cleaved by ubiquitous intracellular furin-like proteases, and the F protein of nonpathogenic viruses is cleaved only by trypsin-like proteases secreted from a limited number of tissues (12).…”

Section: Vol 78 2004 Role Of Hn Protein In Ndv Pathogenesis 4181mentioning

confidence: 99%

The Hemagglutinin-Neuraminidase Protein of Newcastle Disease Virus Determines Tropism and Virulence

Huang

Panda

Elankumaran

et al. 2004

197

164

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.Newcastle disease virus (NDV), the only member of the genus Avulavirus, belongs to the family Paramyxoviridae (25). NDV is an important pathogen of many species of birds; it invokes trade barriers and causes significant economic losses in the commercial poultry industry worldwide. NDV isolates display a spectrum of virulence in chickens, from a fatal to an inapparent infection (1). Strains of NDV are classified into three major pathotypes, depending on the severity of disease produced in chickens. Avirulent strains are termed lentogenic, intermediately virulent strains are termed mesogenic, and highly virulent strains are termed velogenic.The surfaces of NDV particles contain two important functional glycoproteins: the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. In general, membrane glycoproteins drive the assembly and budding of enveloped RNA viruses (41) and are the key players in determining host range and tissue tropism. The F protein mediates both virus-cell and cell-cell fusion (14). The F protein is synthesized as a nonfusogenic precursor, F0, and becomes fusogenic only after cleavage by host cell proteases into disulfide-linked F1 and F2 polypeptides (36). The cleavability of F protein is directly related to the virulence of viruses in vivo. A high content of basic amino acid residues at the F0 cleavage site is correlated with virulence (3, 47). Recent studies with recombinant NDV generated by reverse-genetics techniques showed that modification of a lentogenic F cleavage site to a velogenic cleavage site increased the virulence of the strain (32, 33) but did not reach the vi...