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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 28, 2005 as doi:10.1096/fj.04-2108fje. |
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* INSERM U369, Faculté de Médecine Lyon-R.T.H. Laennec, Lyon, France;
Laboratoire de Virologie, Hospices Civils de Lyon, Domaine Rockefeller, Lyon, France;
Laboratoire NMDA, UMR 6156, Marseille, France;
Idéalp’ Pharma, CEI Lyon-1, Villeurbanne, France; and
|| INSERM U499, Faculté de Médecine Lyon-R.T.H. Laennec, Lyon, France
1 Correspondence: E-mail: greco{at}cgmc.univ-lyon1.fr
SPECIFIC AIMS
All of the conventional antiherpetic drugs available at present exhibit action through inhibition of viral proteins activities. Their extensive clinical use in certain circumstances has led to the emergence of resistant viral strains. An alternative to this traditional drug design approach is to target cellular proteins (Fig. 1
). The aim of this study was to identify cellular proteins that could play a major role in the outcome of infection of cells with wild-type herpes simplex virus type 1 (HSV-1) but, more important, with HSV-1 resistant to conventional drugs.
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PRINCIPAL FINDINGS
HSV-1 protein synthesis is concomitant to the shutoff of synthesis of the major part of host proteins. We have speculated that cellular proteins that escape to this inhibition could determine the outcome of infection. Since spermidine (SPD) and spermine (SPM) are present in large amounts in the envelope and in the nucleocapsid, respectively, of the mature virion, we have investigated whether activity of key enzymes of the polyamine metabolic pathway is required for infection.
1. Inhibition of SAMDC activity prevents HSV-1 infection
S-adenosyl methionine decarboxylase (SAMDC) and ornithine decarboxylase (ODC) are the two key enzymes of the polyamine biosynthetic pathway. Inhibition of SAMDC activity by methylglyoxal bis(guanylhydrazone) (MGBG) efficiently prevented infection: in MGBG-treated cells there was a significant decrease in the level of production of the viral DNA, and of the ICP27 immediate-early, UL42 early, and Us11 late viral mRNAs, together with the corresponding proteins (Fig. 2
, lanes 1, 2). Inhibition of infection was MGBG dose dependent and more efficient in cells pre-exposed to MGBG. In contrast, inhibition of ODC by D,L-2-(difluoromethyl)ornithine only gave rise to a slight inhibition of infection, even at high concentrations.
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2. Treatment of cells with MGBG decreases the amount of intracellular SPM
Mock-infected cells and infected cells were treated for 12–24 h with 100 µM MGBG. There was a 35–40% decrease of the intracellular SPM content in treated cells compared with untreated cells.
3. SAMDC gene expression is specifically stimulated during infection
SAMDC mRNAs were quantified and their distribution among ribosomal fractions was analyzed during infection. The amount of SAMDC mRNA increased progressively until 9 h p.i. while that of the control of ß actin mRNA were progressively decreased. During the course of infection, SAMDC mRNAs were shifted from monoribosomes in mock-infected cells to polyribosomes containing 2–3 ribosomes per mRNA molecule in cells infected for 6 h, and more than 3 ribosomes per mRNA molecule in cells infected for 9 h.
4. MGBG precludes viral infection even when added after the entry step
To determine more precisely whether inhibition takes place after the adsorption step, cells were treated with MGBG either from the beginning of infection, or after the adsorption or penetration step of the virus. Infection was totally prevented when MGBG was added at the beginning of infection, but also when added 2, 4, and 6 h p.i.
5. Addition of exogenous SPM precludes the MGBG-induced inhibition of infection
To verify whether infection depends on the amount of intracellular polyamines, exogenous SPM was added to infected cells treated with MGBG. In the presence of MGBG alone, the amounts of viral DNA and mRNAs produced represented 2–5% of that of the control untreated infected cells, while in cells exposed to both MGBG and SPM, their amounts were 43 and 68% of the control, respectively (Fig. 2)
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6. MGBG inhibits infection after viral entry during the first round of infection
To determine more precisely at which stage of the lytic viral cycle the inhibition of infection takes place, we analyzed viral proteins and DNA during infection of cells exposed or not exposed to MGBG. During the first round of the viral cycle, the amount of the immediate-early viral protein ICP27 was not modified upon treatment. In contrast, the amount of the early UL42 and late Us11 viral proteins and of the viral DNA were decreased.
7. Inhibition of polyamine synthesis prevents infection with HSV-1 strains resistant to acyclovir (ACV) and foscarnet
We tested three HSV-1 reference strains: ACV-susceptible reference strain SC16,ACV-resistant reference strain DM21, and ACV and foscarnet-resistant reference strain TP2.5 (Table 1
). Infection of cells with all the strains presented the same susceptibility to MGBG, even though the virus was resistant to ACV, or resistant to both ACV and foscarnet. MGBG also inhibited replication of HSV-1 clinical isolates resistant to ACV or foscarnet.
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CONCLUSIONS AND SIGNIFICANCE
Although polyamines are present in mature HSV-1 particles, there was no clear link between the polyamine metabolic pathway and the viral replication. In this study, we clearly show that MGBG-induced inhibition of SAMDC activity, a key enzyme of the polyamine synthesis pathway, prevents HSV-1 infection and the production of progeny infectious viruses in a MGBG dose-dependent way. We have investigated the regulation of SAMDC gene expression during HSV-1 infection. We show that SAMDC gene expression is stimulated during infection, contrary to that of the majority of host genes. Upon infection, SAMDC mRNA level increases progressively and SAMDC mRNAs are redistributed from polyribosomes of small size into larger polyribosomes, indicating that SAMDC mRNAs are more efficiently translated after infection than before infection. The very few cellular mRNA that behave in this way usually encode proteins whose synthesis is maintained after infection. These results indicate that SAMDC gene expression is up-regulated in infected cells, and argue for a role of this active polyamine metabolic pathway during viral replication, likely by maintaining a sufficient amount of intracellular polyamines in infected cells.
Our results reveal that MGBG-induced inhibition of infection is due to the decreased amount of available intracellular polyamine, and not to competition among MGBG, polyamines, and viral particles to enter into the cells. As soon as the viral DNA was detected during the first round of infection, its amount was lower in MGBG-treated cells than in untreated infected cells, even when MGBG was added after the viral entry step. The inhibition of infection takes place during the first round of infection, after the beginning of the synthesis of immediate-early proteins and before or during the viral DNA replication.
We demonstrated that the inhibition of SAMDC activity by MGBG also leads to the repression of replication of both HSV-1 laboratory mutant strains and clinical mutant isolates that are resistant to conventional antiviral drugs (e.g., ACV and foscarnet that inhibit the viral thymidine kinase and/or DNA polymerase). The appearance of drug-resistant variants that often emerge from treated patients in response to drug therapy has become a major problem. This is the case for patients compromised by immunosuppressive therapy or after infection by HIV. Therefore, the development of an antiviral strategy targeting a cellular component necessary for the viral replication rather than a viral component makes it possible to avoid emergence of drug-resistant viruses. The inhibition of viral infection is more complete when MGBG and ACV are used in combination rather than alone (data not shown), indicating that the two drugs act through distinct mechanisms. One can expect that in the future, the alternative use of these latter drugs together with conventional drugs for the management of herpetic infections could reduce the appearance and dissemination of drug-resistant viruses.
FOOTNOTES
2 Current address: Centre de Génétique Moléculaire et Cellulaire, UMR 5534 CNRS/UCBL, 43 boulevard du 11 novembre 1918, Villeurbanne 69622, France 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2108fje; doi: 10.1096/fj.04-2108fje
This article has been cited by other articles:
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  A. Calle, I. Ugrinova, A. L. Epstein, P. Bouvet, J.-J. Diaz, and A. Greco Nucleolin Is Required for an Efficient Herpes Simplex Virus Type 1 Infection J. Virol., May 15, 2008; 82(10): 4762 - 4773. [Abstract] [Full Text] [PDF]
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