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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 does not inhibit activity of the HSV-1 protease. The coding sequence of the full-length protein product of UL26 ORF (pUL26) and the expression proteins employed are diagramed in panel (a). Transfection vectors were constructed to express the VP24 protease with an N-terminal HA tag (“protease”), the pUL26.5 with an N-terminal FLAG tag (Substrate 1), or the S129A pUL26 mutant inactive protease-scaffold protein with an N-terminal FLAG tag (Substrate 2). Tags are depicted in grey at the left end of each construct. The release (R) and maturation (M) cleavage sites are shown. After transfection into HEK293-T cells, construct expression and substrate cleavage were evaluated by Western blot using anti-HA and FLAG antibodies. Each protein expressed alone was of the expected size (28.8, 37.9, and 70.6 kD, resp., including tag). Similarly, coexpression of protease with each of the 2 substrates resulted in the N-terminal cleavage products of the expected sizes. The generation of substrate cleavage products was not affected by the addition of NFV at concentrations that inhibit HSV-1 replication in vitro.
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fig2: NFV does not inhibit activity of the HSV-1 protease. The coding sequence of the full-length protein product of UL26 ORF (pUL26) and the expression proteins employed are diagramed in panel (a). Transfection vectors were constructed to express the VP24 protease with an N-terminal HA tag (“protease”), the pUL26.5 with an N-terminal FLAG tag (Substrate 1), or the S129A pUL26 mutant inactive protease-scaffold protein with an N-terminal FLAG tag (Substrate 2). Tags are depicted in grey at the left end of each construct. The release (R) and maturation (M) cleavage sites are shown. After transfection into HEK293-T cells, construct expression and substrate cleavage were evaluated by Western blot using anti-HA and FLAG antibodies. Each protein expressed alone was of the expected size (28.8, 37.9, and 70.6 kD, resp., including tag). Similarly, coexpression of protease with each of the 2 substrates resulted in the N-terminal cleavage products of the expected sizes. The generation of substrate cleavage products was not affected by the addition of NFV at concentrations that inhibit HSV-1 replication in vitro.

Mentions: To determine if NFV, an aspartyl protease inhibitor, could act on the essential HSV-1 UL26 serine protease, we tested whether NFV could inhibit the activity of the HSV-1 protease on its scaffold protein substrates using a cotransfection assay. At NFV concentrations that potently block production of infectious HSV-1, there was no effect on the activity of the HSV-1 protease expressed in HEK293T cells (Figure 2).


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 does not inhibit activity of the HSV-1 protease. The coding sequence of the full-length protein product of UL26 ORF (pUL26) and the expression proteins employed are diagramed in panel (a). Transfection vectors were constructed to express the VP24 protease with an N-terminal HA tag (“protease”), the pUL26.5 with an N-terminal FLAG tag (Substrate 1), or the S129A pUL26 mutant inactive protease-scaffold protein with an N-terminal FLAG tag (Substrate 2). Tags are depicted in grey at the left end of each construct. The release (R) and maturation (M) cleavage sites are shown. After transfection into HEK293-T cells, construct expression and substrate cleavage were evaluated by Western blot using anti-HA and FLAG antibodies. Each protein expressed alone was of the expected size (28.8, 37.9, and 70.6 kD, resp., including tag). Similarly, coexpression of protease with each of the 2 substrates resulted in the N-terminal cleavage products of the expected sizes. The generation of substrate cleavage products was not affected by the addition of NFV at concentrations that inhibit HSV-1 replication in vitro.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4325974&req=5

fig2: NFV does not inhibit activity of the HSV-1 protease. The coding sequence of the full-length protein product of UL26 ORF (pUL26) and the expression proteins employed are diagramed in panel (a). Transfection vectors were constructed to express the VP24 protease with an N-terminal HA tag (“protease”), the pUL26.5 with an N-terminal FLAG tag (Substrate 1), or the S129A pUL26 mutant inactive protease-scaffold protein with an N-terminal FLAG tag (Substrate 2). Tags are depicted in grey at the left end of each construct. The release (R) and maturation (M) cleavage sites are shown. After transfection into HEK293-T cells, construct expression and substrate cleavage were evaluated by Western blot using anti-HA and FLAG antibodies. Each protein expressed alone was of the expected size (28.8, 37.9, and 70.6 kD, resp., including tag). Similarly, coexpression of protease with each of the 2 substrates resulted in the N-terminal cleavage products of the expected sizes. The generation of substrate cleavage products was not affected by the addition of NFV at concentrations that inhibit HSV-1 replication in vitro.
Mentions: To determine if NFV, an aspartyl protease inhibitor, could act on the essential HSV-1 UL26 serine protease, we tested whether NFV could inhibit the activity of the HSV-1 protease on its scaffold protein substrates using a cotransfection assay. At NFV concentrations that potently block production of infectious HSV-1, there was no effect on the activity of the HSV-1 protease expressed in HEK293T cells (Figure 2).

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