INHIBITION OF MAYARO VIRUS REPLICATION BY PROSTAGLANDIN A1 IN Aedes albopictus CELLS

INHIBITION OF MAYARO VIRUS REPLICATION BY PROSTAGLANDIN A₁ IN AEDES ALBOPICTUS CELLS

Joel Antonio Barbosa¹ and Moacyr Alcoforado Rebello^2*

Instituto de Biofísica Carlos Chagas Filho¹ and Departamento de Virologia do Instituto de Microbiologia Prof. Paulo de Góes², Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.

Approved: July 23, 1998

ABSTRACT

Prostaglandin A₁ (PGA₁) inhibits Mayaro virus replication in Aedes albopictus cells at nontoxic doses to uninfected cells. At 10 µg/ml, PGA₁ decreases virus production by 90%. The presence of PGA₁ during virus adsorption, with no treatment after infection, reduces virus yield by 41%. Antiviral activity is observed even when treatment starts at one or two hours post-infection. However, in cells pre-treated with PGA₁ during 24 hours, virus replication is not impaired. Thus, events ocurring during initial stages of infection and after virus adsorption and penetration must be the target of PGA₁ action. SDS-PAGE analysis of ³⁵S-methionine labelled proteins shows that PGA₁ inhibits the synthesis of viral proteins and induces the synthesis of polypeptides with molecular weight of 70 kDa, 57 kDa and 23 kDa. In cells pre-treated with actinomycin D the induction of those proteins is suppressed. In addition, actinomycin D treatment prevents PGA₁ antiviral activity, indicating that PGA₁-induced stress proteins are probably involved in this mechanism.

Key words: Mayaro virus; Prostaglandin; Aedes albopictus cells; Heat-shock proteins; Actinomycin D

Prostaglandins of the A and J type, characterized by the presence of an a, b unsaturated carbonyl group in the cyclopentane ring, have been shown to inhibit the replication of several DNA and RNA virus. However, the antiviral mechanism is not yet well understood and contrasting results have been reported for different virus-cell systems (31).

Several PGs were found to induce heat shock proteins (29, 31). PGA

₁₂

Mayaro virus (Alphavirus genus, Togaviridae family) is an arthropod-borne virus antigenically related to Semliki Forest Virus (8). Mayaro virus, like other Togaviridae, is perpetuated in nature by its ability to infect and replicate both in vertebrate and invertebrate cells (6). In Brazil, this virus has been isolated from human and other mammalian species in the Amazon region. Clinical manifestations of human infection were described as a feverish illness, followed by headache, epigastric pain, backache, chills, nausea and photophobia (8, 3, 9).

We have been studying the Mayaro virus replication in mammalian cells and in mosquito (Aedes albopictus) cells (15, 16). The replication of Mayaro virus in Vero cells is concomitant with an inhibition of host macromolecular synthesis and cythopatic effects. In contrast infection of Aedes albopictus cells with Mayaro virus is characterized by a persistent infection.

Prostaglandins and other active derivatives of polyunsaturated fatty acids have been detected in a large number of invertebrate species (33, 34). Recently, Petzel et al. (21) described the presence of arachidonic acid and prostaglandin E

Aedes aegypti.

We found recently that in PGA

₁Aedes albopictusAedes albopictus₁Aedes albopictus

In the present paper we studied the effect PGA

₁Aedes albopictus₁Cells and virus

Aedes albopictus cells, clone C6/36, were used in this study (12). This cell line was a gift from Dr. R.E. Shope, Arbovirus Unit, Yale University, New Haven, CT, USA. The cells were grown in 60cm

^2o₃^7o

Prostaglandin A

₁^o

Virus infection

Aedes albopictus cells were infected with 1 or 5 PFU per cell in medium without serum. After 60 minutes at 28

^oo₂

Virus titrations were performed by plaque assay in Vero cells (25). Briefly, virus dilutions (0.5 ml) were added to cell monolayers (60 mm diameter Petri dishes) that had just reached confluency. After 60 min at 37

^o₂^oAnalysis of [³⁵S]methionine labelled proteins by polyacrylamide gel electrophoresis

Proteins were labelled with [³⁵S]methionine (20 µCi/ml) and monolayers were directly resuspended in 70 µl of loading buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 5% 2-mercaptoethanol and 0.001% bromophenol blue). Samples were then heated for 5 min at 95^oC and subjected to electrophoresis on one-dimensional 12.5% polyacrylamide gels, using the SDS buffer system of Laemmli (13) at room temperature. The dried gels were exposed to Kodak X-Omat (YAR-S) film. The molecular mass of proteins was determined by co-electrophoresis of standard proteins (Pharmacia). Molecular masses in KDa are indicated to the right (viral proteins) or to the left (cellular proteins) of the gel.

RESULTS

Effect of PGA₁ on Mayaro virus replication

Confluent monolayers of A. albopictus cells were infected with Mayaro virus (1 PFU/cell), and treated with different concentrations of PGA₁from 1h after infection onwards. Supernatants were collected 24h p.i. and virus titers (triplicate samples) were determined by plaque assay. Fig. 1 shows that PGA₁ inhibits virus replication in a dose-dependent manner, with concentrations of 1.0µg/ml being able to reduce virus yield by 50% and doses between 7.5 and 10 µg/ml being effective in causing a 90-95% inhibition of virus production. The concentration of 10µg/ml was found to be the most active nontoxic dose, not having altered cell viability in uninfected cells and was used in all the following experiments. The effect of PGA₁ treatment during virus adsorption was then studied. Cells were infected with Mayaro virus suspension containing PGA₁ (10 µg/ml) or control diluent. After 1h adsortion at 28^oC, virus inocula were removed and the monolayers were washed three times with PBS to remove PGA₁. Growth medium was added and cells were incubated for 24h. The presence of PGA₁ during virus adsorption was found to decrease the number of virus plaques by 41 % (Table 1 protocol A). The antiviral action of PGA₁ was observed even when the treatment started 1 or 2h p.i. (Table 1 protocol B and C). However, in cells pre-treated with PGA₁during 24h (with no treatment after infection) virus replication was not impaired (protocols D and E).

Figure 1
- Effect of PGA₁ on Mayaro virus production. Dose-dependent inhibition. A. albopictus cells were infected with Mayaro virus (1 pfu/ml) and treated with different concentrations of PGA₁. Virus production was determined (triplicate samples) 24 hours post-infection as described in Materials and Methods.

Table 1
- Inhibition of Mayaro virus in A. albopictus cells by PGA₁ treatment. Virus yield of controls was 6,7 x 10⁷ PFU/ml.

Cells were infected with Mayaro virus (1 PFU/cell) and treated with PGA₁ (10 µg/ml). Virus yields were collected 24 hours post-infection and titrated (triplicate samples) as described in Material and Methods.

Protocol A: PGA₁ treatment only during virus adsorption period. Protocol B : PGA₁ treatment started 1 hour after virus adsorption period. Protocol C: PGA₁ treatment started 2 hours after virus adsorption period. Protocol D : Cells were pre-treated with PGA₁ during 24 hours. After this period, monolayers were washed with PBS, infected with Mayaro virus and incubated with growth medium in the presence of PGA₁. Twenty four hours later, the virus were titrated. Protocol E : Cells were pre-treated with PGA₁ during 24 hours. After this period, monolayers were washed with PBS, infected with Mayaro virus and incubated in growth medium. Twenty four hours later, the virus were titrated.

Effect of PGA₁ on protein synthesis

Previous results from our laboratory (Mezencio and Rebello, 1993) showed that in Mayaro virus infected BHK-21 cells and in A. albopictus cells, six viral proteins could be detected. Three of them are structural proteins (molecular weight of 54kDa, 50 kDa and 34 kDa,) and the other three are precursors of viral proteins (molecular weight of 110 kDa, 64kDa and 62kDa).

To examine the effect of PGA₁ on the protein synthesis of Mayaro virus infected A. albopictus cells, confluent monolayers were infected (5 PFU/ml) and 1h later, virus inocula were removed and culture medium containing PGA₁ or control diluent was added. After 24h, the medium was supplemented with 20 µCi/ml of ³⁵S-methionine. One hour later, the medium was removed and the proteins were analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 2, treatment with PGA₁ suppressed viral protein synthesis. The synthesis of protein p34 was dramatically reduced in cells treated with PGA₁ (lanes C and D) and the synthesis of the two precursors (p64 and p62) was also modified (lane D). We also observed that in mock infected cells, the presence of PGA₁ stimulated the synthesis of three polypeptides (stress proteins) with molecular weights of 70kDa, 57kDa and 23kDa (lane B).

Figure 2
- Effect of PGA₁ on Mayaro virus infected cells. A. albopictus cells were infected (lanes C and D) with Mayaro virus (5 pfu/ml) or mock infected (lanes A and B) and maintained in growth medium for 24 hours in the presence of PGA₁ (lanes B and D). After this period, the cells were labelled with ³⁵S-methionine (20 µCi/ml) for 1 hour and cellular extracts were subjected to polyacrylamide gel electrophoresis.

Effect of Actinomycin D (AMD) and PGA₁ on synthesis of Mayaro virus protein

In Fig. 3 we show that, in cells treated with AMD (2 µg/ml) the induction of stress proteins was suppressed (lane B). In A. albopictus infected cells, we observed that in the presence of AMD there was only a partial reduction on viral protein synthesis, probably as a consequence of the long treatment with this drug (lane D). In cells treated with PGA

₁₁

Figure 3
- Effect of PGA₁ and actinomycin D in Mayaro virus protein synthesis. A albopictus cells were infected (lanes C, D, E and F) with Mayaro virus (5pfu/ml) or mock infected (lanes A and B) and maintained in growth medium for 24 hours, in the presence of PGA₁ (lanes A, B, E and F) and actinomycin D (lanes B, D and F). After this period the cells were labeled with ³⁵S-methionine (20 µCi/ml) for 1 hour and cellular extracts were subjected to polyacrylamide gel electrophoresis.

₁A. albopictus₁

The presence of PGA

₁₁et al.

The pre-treatment of A. albopictus cells with PGA

₁et al.₁

The examination of viral proteins revealed that PGA

₁et al.₁₀et al.₂

Recent results from experiments concerning the effects of PGJ

₂³

Actinomycin D blocked the PGA

₁₁₁^o₁₁

On the other hand, Parker et al. (20) observed that in mouse L929 cells actinomycin D inhibited cellular RNA synthesis but failed to block the antiviral activity of PGA

₁

The results presented in this paper show that in cultured A. albopictus cells, PGA

₁et al.et al.

RESUMO

Inibição da replicação do vírus Mayaro pela prostaglandin A₁ em células de Aedes albopictus

Prostaglandin A₁ (PGA₁) inibe a replicação do vírus Mayaro em células de Aedes albopictus em doses não tóxicas para células não infectadas. A presença de PGA₁ durante o período de adsorção, reduz a produção do vírus em 41%. A atividade antiviral é observada mesmo quando o tratamento é iniciado com 1 ou 2 horas após a infecção. Entretanto, em células pré-tratadas com PGA₁ durante 24 h a replicação viral é inalterada. A análise das proteínas marcadas com ³⁵S-metionina, por eletroforese em gel de poliacrilamida, mostra que a PGA₁ inibe a síntese de proteínas virais e induz a síntese de polipeptídeos com peso molecular de 70 Kda, 57 Kda e 23 Kda. Em células pré-tratadas com actinomicina D observa-se um bloqueio da atividade antiviral, indicando que as proteínas de estresse induzidas pela PGA₁ estão envolvidas neste mecanismo.

Palavras-chave: Vírus Mayaro; Prostaglandina A₁; Células de Aedes albopictus; Proteínas de Choque Térmico, Actinomicina D.

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*

Corresponding author. Mailing address: Departamento de Virologia, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, RJ, Brasil. Telefax: ([phone] / 270-8793

Publication Dates

• Publication in this collection
  26 Feb 1999
• Date of issue
  Sept 1998

History

• Accepted
  23 July 1998
• Reviewed
  17 Apr 1998
• Received
  09 Feb 1998