Nelfinavir inhibits maturation and export of herpes simplex virus 1

Abstract

Nelfinavir (NFV) is an HIV-1 protease inhibitor with demonstrated antiviral activity against herpes simplex virus 1 (HSV-1) and several other herpesviruses. However, the stages of HSV-1 replication inhibited by NFV have not been explored. In this study, we investigated the effects of NFV on capsid assembly and envelopment. We confirmed the inhibitory effects of NFV on HSV-1 replication by plaque assay and found that treatment with NFV did not affect capsid assembly, activity of the HSV-1 maturational protease, or formation of DNA-containing capsids in the nucleus. Confocal and electron microscopy studies showed that these capsids were transported to the cytoplasm but failed to complete secondary envelopment and subsequent exit from the cell. Consistent with the microscopy results, a light-scattering band corresponding to enveloped virions was not evident following sucrose gradient rate-velocity separation of lysates from drug-treated cells. Evidence of a possibly related effect of NFV on viral glycoprotein maturation was also discovered. NFV also inhibited the replication of an HSV-1 thymidine kinase mutant resistant to nucleoside analogues such as acyclovir. Given that NFV is neither a nucleoside mimic nor a known inhibitor of nucleic acid synthesis, this was expected and suggests its potential as a coinhibitor or alternate antiviral therapeutic agent in cases of resistance.

Importance: Nelfinavir (NFV) is a clinically important antiviral drug that inhibits production of infectious HIV. It was reported to inhibit herpesviruses in cell culture. Herpes simplex virus 1 (HSV-1) infections are common and often associated with several diseases. The studies we describe here confirm and extend earlier findings by investigating how NFV interferes with HSV-1 replication. We show that early steps in virus formation (e.g., assembly of DNA-containing capsids in the nucleus and their movement into the cytoplasm) appear to be unaffected by NFV, whereas later steps (e.g., final envelopment in the cytoplasm and release of infectious virus from the cell) are severely restricted by the drug. Our findings provide the first insight into how NFV inhibits HSV-1 replication and suggest that this drug may have applications for studying the herpesvirus envelopment process. Additionally, NFV may have therapeutic value alone or in combination with other antivirals in treating herpesvirus infections.

FIG 1

HSV-1 UL26 protease expression and activity are not affected by nelfinavir. Shown here is an immunoblot of Vero cells infected with wild-type virus (KOS) or protease-inactive mutant virus (KUL26H61E) that had been incubated for 24 h in the absence (−) or presence (+) of 10 μM NFV. Blots were probed with antibodies against the amino end of pUL26, stripped, and reprobed with antibodies to actin. Antibodies were detected by enhanced chemiluminescence and exposure to Kodak film. Abbreviations indicate full-length UL26 protease (pUL26) and its cleavage product, VP24. Marker protein sizes (kDa) are shown between images. HSV-1 UL26 protease expression and activity were not affected by nelfinavir.

FIG 2

NFV does not alter HSV capsid production. Vero cells were infected at an MOI of 10, incubated with either no drug (control) or 10 μM NFV, and metabolically labeled with [³⁵S]methionine from 9 to 24 hpi. (A) At 24 hpi, detergent-treated whole-cell extracts were prepared and loaded onto 20 to 50% sucrose gradients. Digital pictures of the gradients show light-scattering bands corresponding to the sedimentation of A, B, and C capsids. (B) Twelve sequential fractions were collected from both gradients, and radioactivity in each fraction was measured by liquid scintillation. Corresponding peaks of radioactivity for C capsids (fraction 5), B capsids (fraction 8), and A capsids (fraction 10) were observed in the two gradients. (C) The proteins in fractions 5, 8, and 10 (C, B, and A capsids) from both gradients were precipitated with trichloroacetic acid and analyzed by SDS-PAGE. Shown here is a fluorogram of the resulting gel. Viral proteins are indicated on the right, and molecular weight markers are indicated on the left.

FIG 3

Tegument and glycoprotein differences detected between NFV-treated and nontreated HSV-infected cells. (A) Material was collected from the virion position of gradients containing detergent-free lysates of cells cultured with [³⁵S]methionine, with or without 10 μM NFV, and subjected to SDS-PAGE. Shown here is a fluorogram of the resulting gel. Representative capsid (VP5 and VP26), tegument (VP1,2), and envelope (gB/gC and gD) proteins are indicated. (B) Radiolabeled glycoproteins were immunoprecipitated from NFV-treated (+; 10 μM NFV) and nontreated (−) infected cells. Antisera used are indicated at the top. Dots in panel B denote fully glycosylated (mature) forms of the glycoproteins; asterisks denote proteins with electrophoretic mobilities consistent with immature gB, gC, and gD in NFV-treated cells. Proteins corresponding to immature forms of the glycoproteins are similarly indicated in panel A.

FIG 4

Nelfinavir inhibits maturation and export of virus. (A) Confocal (GFP) panels. Vero cells were infected with a GFP-expressing recombinant virus (K26GFP), incubated without (No NFV) or with 10 μM NFV for 16 h, and examined in live culture by confocal microscopy. Red arrowheads indicate viral nuclear replication sites, white arrowheads indicate the nuclear membrane, and the white arrow indicates the plasma membrane. Insets show merged GFP and phase-contrast images of single cells in nontreated and NFV-treated cells, with the nuclear (N; fluorescent in both) and plasma membranes (P; fluorescent only in nontreated cells) indicated. (B) EM (cell) panels. Vero cells infected with wild-type virus were incubated for 16 h without (No NFV) or with 10 μM NFV and then processed and evaluated by electron microscopy. Black arrowheads indicate DNA-filled capsids in the nucleus (N), white arrowheads indicate enveloped or nonenveloped capsids in the cytoplasm (C), and the white arrow indicates mature virions in the extracellular space of nontreated cells. Bars = 400 nm. (C) EM (cytoplasm) panels. Enlarged cytoplasmic views are shown. The thick arrow at the bottom indicates an enveloped capsid in a nontreated cell; thin arrows indicate nonenveloped capsids present in both nontreated and treated cells. Bars = 200 nm. Particle counts were as follows: for nuclear capsids (B), ∼20 without NFV and ∼20 with NFV; for enveloped nucleocapsids outside the cell (B), ∼16 without NFV and 0 with NFV; for enveloped capsids in cytoplasm (C), ∼15 without NFV and 0 with NFV; and for nonenveloped capsids in cytoplasm (C), ∼12 without NFV and ∼30 with NFV.

FIG 5

Absence of viral TK does not affect antiviral activity of NFV. Cells infected with either wild-type (KOS) or TK mutant (dlsactk) virus at an MOI of 10 were incubated without drug (DMSO), with ganciclovir (100 μM GCV), or with 10 μM NFV for 24 h. Virus yields were determined by titration on Vero cells. Results are means ± standard errors of the means (SEM) for two independent experiments.