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Nelfinavir impairs glycosylation of herpes simplex virus 1 envelope proteins and blocks virus maturation.

Gantt S, Gachelet E, Carlsson J, Barcy S, Casper C, Lagunoff M - Adv Virol (2015)

Bottom Line: NFV did not significantly affect the level of expression of late HSV-1 gene products.NFV was found to decrease glycosylation of viral glycoproteins B and C and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by NFV.These results demonstrate that NFV causes alterations in HSV-1 glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for HHV replication.

View Article: PubMed Central - PubMed

Affiliation: Seattle Children's Research Institute, University of Washington, Seattle, WA 98101, USA ; Department of Pediatrics, University of Washington, Seattle, WA 98105, USA ; Department of Global Health, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Nelfinavir (NFV) is an HIV-1 aspartyl protease inhibitor that has numerous effects on human cells, which impart attractive antitumor properties. NFV has also been shown to have in vitro inhibitory activity against human herpesviruses (HHVs). Given the apparent absence of an aspartyl protease encoded by HHVs, we investigated the mechanism of action of NFV herpes simplex virus type 1 (HSV-1) in cultured cells. Selection of HSV-1 resistance to NFV was not achieved despite multiple passages under drug pressure. NFV did not significantly affect the level of expression of late HSV-1 gene products. Normal numbers of viral particles appeared to be produced in NFV-treated cells by electron microscopy but remain within the cytoplasm more often than controls. NFV did not inhibit the activity of the HSV-1 serine protease nor could its antiviral activity be attributed to inhibition of Akt phosphorylation. NFV was found to decrease glycosylation of viral glycoproteins B and C and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by NFV. These results demonstrate that NFV causes alterations in HSV-1 glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for HHV replication.

No MeSH data available.


Related in: MedlinePlus

NFV affects the glycosylation and subcellular localization of viral gene products but does not change the level of expression. (a) Western blots of HSV-1 infected HF cells show increased mobility in gB and gC with NFV treatment, compared to cells that were untreated or treated with indinavir (IDV). Eastern blots show that altered staining by lectins (peanut agglutinin, PNA; ricinus communis agglutinin I, RCA-I; wheat germ agglutinin, WGA; and concanavalin A, ConA) demonstrates reduced overall glycosylation in NFV-treated cells. (b) HSV-1 gB localization (red staining) is altered in NFV-treated HF cells, showing decreased delineation of plasma membrane processes (indicated by white arrows) by IFA compared to untreated cells. Nuclei are stained blue.
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fig5: NFV affects the glycosylation and subcellular localization of viral gene products but does not change the level of expression. (a) Western blots of HSV-1 infected HF cells show increased mobility in gB and gC with NFV treatment, compared to cells that were untreated or treated with indinavir (IDV). Eastern blots show that altered staining by lectins (peanut agglutinin, PNA; ricinus communis agglutinin I, RCA-I; wheat germ agglutinin, WGA; and concanavalin A, ConA) demonstrates reduced overall glycosylation in NFV-treated cells. (b) HSV-1 gB localization (red staining) is altered in NFV-treated HF cells, showing decreased delineation of plasma membrane processes (indicated by white arrows) by IFA compared to untreated cells. Nuclei are stained blue.

Mentions: During evaluation of HSV-1 glycoproteins gB and gC expression by Western blotting, it was apparent that NFV treatment of infected cells resulted in increased electrophoretic mobility (Figure 5(a)) compared to untreated controls or IDV-treated cells. The apparent change in molecular weight of these viral proteins was estimated to be consistent with the reduction in glycosylation [30–32]. To assess the effect of NFV on protein glycosylation in HSV-1-infected cells, Eastern blotting was performed using lectins PNA, RCA-I, WGA, and ConA (Figure 5(a)). Compared to untreated or IDV-treated cells, NFV resulted in a marked reduction in staining by PNA, RCA-I, and WGA indicating decreased addition of galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine [33, 34]. In contrast, ConA staining was not appreciably reduced, suggesting relatively normal levels of oligomannose-type N-glycans. NFV resulted in a clear alteration in the subcellular localization of HSV-1 gB (Figure 5(b)) by immunofluorescent antibody staining. Compared with control treatments in which viral envelope glycoprotein staining uniformly delineated the plasma membrane of HSV-1-infected HF cells, with NFV treatment staining appeared predominantly perinuclear, suggesting improper trafficking to the cell surface. Consistent with the electron microscopy results, using immunofluorescence, no increase in LC3-II staining, a marker of autophagy, was apparent (data not shown).


Nelfinavir impairs glycosylation of herpes simplex virus 1 envelope proteins and blocks virus maturation.

Gantt S, Gachelet E, Carlsson J, Barcy S, Casper C, Lagunoff M - Adv Virol (2015)

NFV affects the glycosylation and subcellular localization of viral gene products but does not change the level of expression. (a) Western blots of HSV-1 infected HF cells show increased mobility in gB and gC with NFV treatment, compared to cells that were untreated or treated with indinavir (IDV). Eastern blots show that altered staining by lectins (peanut agglutinin, PNA; ricinus communis agglutinin I, RCA-I; wheat germ agglutinin, WGA; and concanavalin A, ConA) demonstrates reduced overall glycosylation in NFV-treated cells. (b) HSV-1 gB localization (red staining) is altered in NFV-treated HF cells, showing decreased delineation of plasma membrane processes (indicated by white arrows) by IFA compared to untreated cells. Nuclei are stained blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4325974&req=5

fig5: NFV affects the glycosylation and subcellular localization of viral gene products but does not change the level of expression. (a) Western blots of HSV-1 infected HF cells show increased mobility in gB and gC with NFV treatment, compared to cells that were untreated or treated with indinavir (IDV). Eastern blots show that altered staining by lectins (peanut agglutinin, PNA; ricinus communis agglutinin I, RCA-I; wheat germ agglutinin, WGA; and concanavalin A, ConA) demonstrates reduced overall glycosylation in NFV-treated cells. (b) HSV-1 gB localization (red staining) is altered in NFV-treated HF cells, showing decreased delineation of plasma membrane processes (indicated by white arrows) by IFA compared to untreated cells. Nuclei are stained blue.
Mentions: During evaluation of HSV-1 glycoproteins gB and gC expression by Western blotting, it was apparent that NFV treatment of infected cells resulted in increased electrophoretic mobility (Figure 5(a)) compared to untreated controls or IDV-treated cells. The apparent change in molecular weight of these viral proteins was estimated to be consistent with the reduction in glycosylation [30–32]. To assess the effect of NFV on protein glycosylation in HSV-1-infected cells, Eastern blotting was performed using lectins PNA, RCA-I, WGA, and ConA (Figure 5(a)). Compared to untreated or IDV-treated cells, NFV resulted in a marked reduction in staining by PNA, RCA-I, and WGA indicating decreased addition of galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine [33, 34]. In contrast, ConA staining was not appreciably reduced, suggesting relatively normal levels of oligomannose-type N-glycans. NFV resulted in a clear alteration in the subcellular localization of HSV-1 gB (Figure 5(b)) by immunofluorescent antibody staining. Compared with control treatments in which viral envelope glycoprotein staining uniformly delineated the plasma membrane of HSV-1-infected HF cells, with NFV treatment staining appeared predominantly perinuclear, suggesting improper trafficking to the cell surface. Consistent with the electron microscopy results, using immunofluorescence, no increase in LC3-II staining, a marker of autophagy, was apparent (data not shown).

Bottom Line: NFV did not significantly affect the level of expression of late HSV-1 gene products.NFV was found to decrease glycosylation of viral glycoproteins B and C and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by NFV.These results demonstrate that NFV causes alterations in HSV-1 glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for HHV replication.

View Article: PubMed Central - PubMed

Affiliation: Seattle Children's Research Institute, University of Washington, Seattle, WA 98101, USA ; Department of Pediatrics, University of Washington, Seattle, WA 98105, USA ; Department of Global Health, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Nelfinavir (NFV) is an HIV-1 aspartyl protease inhibitor that has numerous effects on human cells, which impart attractive antitumor properties. NFV has also been shown to have in vitro inhibitory activity against human herpesviruses (HHVs). Given the apparent absence of an aspartyl protease encoded by HHVs, we investigated the mechanism of action of NFV herpes simplex virus type 1 (HSV-1) in cultured cells. Selection of HSV-1 resistance to NFV was not achieved despite multiple passages under drug pressure. NFV did not significantly affect the level of expression of late HSV-1 gene products. Normal numbers of viral particles appeared to be produced in NFV-treated cells by electron microscopy but remain within the cytoplasm more often than controls. NFV did not inhibit the activity of the HSV-1 serine protease nor could its antiviral activity be attributed to inhibition of Akt phosphorylation. NFV was found to decrease glycosylation of viral glycoproteins B and C and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by NFV. These results demonstrate that NFV causes alterations in HSV-1 glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for HHV replication.

No MeSH data available.


Related in: MedlinePlus