Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model

Lucio Ayres Caldas Davis Fernandes Ferreira Tânia Rosaria Pereira Freitas About the authors

Abstract

INTRODUCTION:

The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment.

METHODS:

Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively.

RESULTS:

PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography.

CONCLUSIONS:

PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

Keywords:
Mayaro virus; Prostaglandin A; Heat shock proteins

INTRODUCTION

Genus Alphavirus includes human and animal pathogens for which no effective antiviral treatment is yet available. This genus includes the Mayaro virus [(MAYV); genus Alphavirus, family Togaviridae], the causative agent of Mayaro fever (MF), a dengue-like disease characterized by acute fever, maculopapular rash, intense arthralgia and nausea11. Vasconcelos PFC, Travassos da Rosa APA, Pinheiro FP, Shope RE, Travassos da Rosa JFS, Rodrigues SG, et al. Arboviruses pathogenic for man in Brazil. In: Travassos da Rosa APA, Vasconcelos PFC, Travassos da Rosa JFS, editors. An overview of arbovirology in Brazil and neighbouring countries. Belém: Instituto Evandro Chagas; 1998. p.72-99.. MAYV is an Arbovirus, transmitted mainly by mosquitoes (Haemagogus spp.), which was first described in Trinidad, in 195422. Anderson CR, Downs WG, Wattley GH, Ahin NW, Reese AA. Mayaro virus: a new human disease agent. II. Isolation from blood of patients in Trinidad, B.W.I. Am J Trop Med Hyg. 1957;6(6):1012-6.. Birds are secondary hosts of MAYV and may spread the infection; this is observed in several countries in Central and South America33. Esposito DLA, Fonseca BALD. Will Mayaro virus be responsible for the next outbreak of an arthropod-borne virus in Brazil? Braz J Infect Dis. 2017;21(5)540-4.. Both acute and chronic phases of MF often present nonspecific symptoms that may confound diagnosis. Joint pain, characteristic of chronic MF, may persist for at least a year after infection. In MAYV endemic areas, patients with joint pain who are seronegative for arthritis may be infected with MAYV44. Pauvolid-Corrêa A, Juliano RS, Campos Z, Velez J, Nogueira RM, Komar N. Neutralising antibodies for Mayaro virus in Pantanal, Brazil. Mem Inst Oswaldo Cruz. 2015;110(1):125-33.,55. Halsey ES, Siles C, Guevara C, Vilcarromero S, Jhonston EJ, Ramal C, et al. Mayaro virus infection, Amazon Basin region, Peru, 2010-2013. Emerg Infect Dis. 2013;19(11):1839-42..

In Brazil, attention first focused on MAYV in 1955, due to an outbreak in the State of Pará66. Causey OR, Maroja OM. Mayaro virus: a new human disease agent. III. Investigation of an epidemic of acute febrile illness on the river Guama in Pará, Brazil and isolation of Mayaro virus as causative agent. Am J Trop Med Hyg . 1957;6(6):1017-23.. Since then, MAYV infections have been detected in several states77. Coimbra TL, Santos CL, Suzuki A, Petrella SM, Bisordi I, Nagamori AH, et al. Mayaro virus: imported cases of human infection in São Paulo State, Brazil. Rev Inst Med Trop Sao Paulo. 2007;49(4):221-4.

8. Azevedo RSS, Silva EVP, Carvalho VL, Rodrigues SG, Nunes Neto JP, Monteiro HAO, et al. Mayaro fever virus, Brazilian Amazon. Emerg Infect Dis . 2009;15(11):1830-2.
-99. Casseb AR, Casseb LMN, Silva SP, Vasconcelos PFC. Arbovírus: importante zoonose na amazônia brasileira. Vet e Zootec. 2013;20(3):391-403.. MAYV is endemic to the Amazon region and Central Brazil, as confirmed by a high incidence of immunoglobulin M (IgM) against MAYV in febrile patients in the Goiás State1010. Brnini S, França DDS, Silva JB, Silva LN, Silva FPA, Spadoni M, et al. High frequency of Mayaro virus IgM among febrile patients, Central Brazil. Emerg Infect Dis . 2017;23(6):1025-6.. Additionally, Terzian et al.1111. Terzian AC, Auguste AJ, Vedovello D, Ferreira MU, da Silva-Nunes M, Sperança MA, et al. Isolation and characterization of Mayaro virus from a human in Acre, Brazil. Am J Trop Med Hyg . 2015;92(2):401-4. isolated a genetically distinct Brazilian MAYV lineage in the State of Acre.

MAYV detection in equids, sheep and caimans provided indirect evidence of MAYV circulation in the Brazilian Western region44. Pauvolid-Corrêa A, Juliano RS, Campos Z, Velez J, Nogueira RM, Komar N. Neutralising antibodies for Mayaro virus in Pantanal, Brazil. Mem Inst Oswaldo Cruz. 2015;110(1):125-33., reinforcing the outbreak potential of this virus, and revealing previously unknown hosts. MAYV infection rates may be underestimated, as MF is commonly mistaken for dengue, in the absence of specific diagnostics1212. Zuchi N, Heinen LBS, dos Santos MAM, Pereira FC, Slhessarenko RD. Molecular detection of Mayaro virus during a dengue outbreak in the state of Mato Grosso, Central-West Brazil. Mem Inst Oswaldo Cruz . 2014;109(6):820-3.. Some cases of a dengue-like viral disease, attributed to yet another alphavirus, the Chikungunya virus (CHIKV), during a recent outbreak in Brazil, may have been caused by MAYV infection1313. Figueiredo ML, Figueiredo LT. Emerging alphaviruses in the Americas: Chikungunya and Mayaro. Rev Soc Bras Med Trop. 2014;47(6):677-83..

Similar to CHIKV, and all other alphaviruses, MAYV genome consists of a single strand of positive-sense RNA, which is 12kb in length and encapsulated in an icosahedral capsid composed of a single protein (the C protein) and enveloped by a lipid bilayer derived from host cell plasma membrane. The genome is arranged in two sections: the 5′ two-thirds encodes non-structural proteins required for transcription and replication of RNA, with the 5′ terminus capped with a 7-methylguanosine; the 3′ one-third encodes structural proteins translated from sub-genomic messenger RNA (mRNA), with a polyadenylated 3′ terminus. Viral replication occurs in host cell cytoplasm, and virions mature by budding through the plasma membrane, where virus-encoded surface glycoproteins E2 and E1 are embedded1414. Johnston RE, Peters CJ. Alphaviruses. In: Fields BN, Knipe DM, Howley PM, eds. Virology. 3rd edition. Philadelphia: Lippincott-Raven Publishers; 1996. p.843-98.,1515. Paul D, Bartenschlager R. Architecture and biogenesis of plus-strand RNA virus replication factories. World J Virol. 2013;2(2):32-48..

Serologically, MAYV is most closely related to the Semliki Forest virus (SFV) and to CHIKV. All three viruses are considered members of the Semliki Forest Complex. Similar to CHIKV, MAYV is transmitted from the adult mosquito to its progeny. Intrinsically high copy error rates of viral RNA polymerases have resulted in an extension of the CHIKV geographical range1616. Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog. 2007;3(12):e201.. This phenomenon may also occur with MAYV. Therefore, the severe public health risk of a MAYV geographical spread due to adaptation to other mosquito species should not be underestimated, as there is currently no vaccine or effective treatment against MF.

This study aimed to increase the understanding of MAYV morphogenesis, by examining MAYV/host cell interactions and the effect of prostaglandin (PG) on viral replication. PGs are naturally occurring cyclic fatty acids that are synthesized from polyunsaturated fatty acid precursors, induced by cell injury and inflammation1717. Funk CD. Prostaglandins and leukotrienes: Advances in eicosanoid biology. Science. 2001;294(5548):1871-5.. PGs play regulatory roles in physiological processes such as eukaryotic cell proliferation, differentiation, inflammation and febrile conditions1717. Funk CD. Prostaglandins and leukotrienes: Advances in eicosanoid biology. Science. 2001;294(5548):1871-5.

18. Dinarello CA, Wolff SM. Molecular basis of fever in humans. Am J Med. 1982;72(5):799-819.
-1919. Rossi A, Kapahi P, Natoli G, Takahashi T, Chen Y, Karin M, et al. Anti-inflamatory cyclopentenone prostaglandins are direct inhibitors of IκB kinase. Nature. 2000;403(6765):103-8.. Antiviral properties of PGs are evident, mainly among A and J type PGs2020. Fukushima M, Kato T, Narumiya S, Mizushima Y, Sasaki H, Terashima Y, et al. Prostaglandin A and J: antitumor and antiviral prostaglandins. Adv Prostaglandin Thromboxane Leukot Res. 1989;19:415-8.. A1 and J2 type PGs are well known for their antiviral effect. Chemical analysis of these PGs indicated that the presence of a carbonyl group at the unsaturated (α, β) cyclopentane ring might be responsible for this property2121. Fukushima M. Prostaglandin J2 - anti-tumour and anti-viral activities and the mechanisms involved. Eicosanoids. 1990;3(4):189-99. Erratum in: Eicosanoids 1991;4(2):119.,2222. Santoro MG. Antiviral activity of cyclopentanone prostanoids. Trends Microbiol. 1997;5(7):276-81.. PGs inhibit production of MAYV2323. Ishimaru D, Marcicano FG, Rebello MA. Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells. Braz J Med Biol Res. 1998;31(9):1119-23. and other viruses, including Sendai2424. Santoro MG, Amici C, Elia G, Benedetto A, Garaci E. Inhibition of virus protein glycosilation as the mechanism of the antiviral action of prostaglandin A in Sendai virus-infected cells. J Gen Virol. 1989;70(Pt 4):789-800., Vesicular stomatitis virus (VSV)2525. Santoro MG, Jaffe BM, Esteban M. Prostaglandin A inhibits the replication of vesicular stomatitis virus: effect on virus glycoprotein. J Gen Virol . 1983;64(Pt 12):2797-801.,2626. Bader T, Ankel H. Inhibition of primary transcription of Vesicular Stomatitis virus by prostaglandin A1. J Gen Virol . 1990;71(Pt 12):2823-32., human immunodeficiency virus (HIV)2727. Ankel H, Turriziani O, Antonelli G. Prostaglandin A inhibits replication of human imunodeficiency virus during acute infection. J Gen Virol . 1991;72(Pt 11):2797-800., Rotavirus2828. Superti F, Amici C, Tinari A, Donelli G, Santoro MG. Inhibition of Rotavirus replication by prostaglandin A: evidence for a block of virus maturation. J Infect Dis. 1998;178(2):564-8. and Classical swine fever virus2929. Freitas TR, Caldas LA, Rebello MA. Prostaglandin A1 inhibits replication of classical swine fever virus. Mem Inst Oswaldo Cruz . 1998;93(6):815-8..

Despite the broad antiviral effects of PGs, mechanisms underlying antiviral activity of PGs, which vary among different virus-cell systems, are not yet understood. Heat shock proteins (HSPs), known for their chaperone function, are induced under PG treatment, representing a potential mechanism underlying antiviral effects of PGs3030. de Marco A, Carattoli A, Rozera C, Fortini D, Giorgi C, Belardo G, et al. Induction of the heat-shock response by antiviral prostaglandins in human cells infected with human immunodeficiency virus type 1. Eur J Biochem. 1998;256(2):334-41.,3131. Sugiyama R, Nishitsuji H, Furukawa A, et al. Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G. J Biol Chem. 2013;286(12):10051-7.. We evaluated the effect of PGA1 on MAYV production, and the relevance of the heat-shock protein, HSP70, to this process.

METHODS

Chemicals

PGA1 was purchased from Sigma Chemical Company, St Louis, USA. It was diluted in ethanol (1mg/mL) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, at 20μg/mL). Both stock solutions were stored at -20°C.

Cell culture and virus

Human cervix carcinoma (Hep-2 cells, ATCC®CCL-23™) were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum, and incubated at 37°C and 5% CO2 in 25cm2 cell culture flasks.

MAYV (lineage TR 4675) was stored at -80°C. The virus was obtained from the American Type Culture Collection (Rockville, MD, USA), and propagated in vitro in Hep-2 cells.

Virus Infection and PGA1 treatment

Hep-2 cell monolayers (~80% confluency) were inoculated with MAYV at a multiplicity of infection (MOI) of 1, for drug effect evaluation using viral titer assays, or 5, for electron microscopy and viral protein analysis. Following the virus adsorption period (1h, at 37°C and 5% CO2), inoculum was removed and fresh medium added. Depending on the assay, cells were pre-treated with PGA1 for 12h prior to infection, or treated with different PGA1 concentrations for different post-infection periods under standard culture conditions (37°C and 5% CO2). At specific treatment times, culture supernatants were collected for virus titer determination via plaque assay, as per Baer & Kehn-Hall3232. Baer A, Kehn-Hall K. Viral concentration determination through plaque assays: using traditional and novel overlay systems. J Vis Exp. 2014;93:e52065..

Cell viability assay

Cell monolayers were incubated in a solution containing 5µg/mL neutral red diluted in phosphate buffered saline (PBS), for 3h at 37°C, in 96-well plates. After discarding the solution, cells were incubated with 4% formaldehyde (in PBS) for 1 min, and 100% methanol for 20 min. Samples were analyzed for absorbance at 490nm, using an enzyme-linked immunosorbent assay (ELISA) plate reader (SpectraMax M2).

Radioactive labelling, SDS-PAGE and densitometry

Cells were infected as described (section 2.2), and treated with PGA1 for different periods of time, following which the culture medium was replaced by DMEM devoid of L-cysteine and L-methionine and containing 30μCi/mL 3535. Pinheiro FP, Freitas RB, Travassos JF, Da Rosa YBG, MelloWA, LeDuc JW. An outbreak of Mayaro virus disease in Belterra, Brazil. I. Clinical and virological findings. Am J Trop Med Hyg .1981;30(3):674-81.S-methionine. After 60 min of incubation, the medium was removed and 80µL of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (25mM Tris-HCl containing 10% glycerol, 0.02% bromophenol blue, 100mM dithiothreitol and 2% SDS) was added to each flask. Samples were heated for 5 min at 100oC, and subjected to SDS-PAGE on a 12% polyacrylamide gel. Radioactivity was detected by exposing gels directly to X-ray films (BioMax TranScreen LE intensifying screen, Eastman Kodak Company, USA). Densitometric analysis of autoradiography films was performed using the Bromma 2202 laser densitometer (LKB-Produkter).

Transmission electron microscopy

Hep-2 monolayers grown in cell culture flasks of 25cm2 (infected and treated as described in section 2.3) were fixed for 2 h at room temperature, in 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2), and post-fixed at room temperature in 1% OsO4/0.8% potassium ferrocyanide in the same buffer for 1h. Samples were dehydrated in ethanol and flat-embedded in Polybed resin (Polysciences®). Ultrathin sections (60nm thick) were stained with uranyl acetate and lead citrate, and observed using a Zeiss 900 transmission electron microscope.

Western blot

Samples subjected to SDS-PAGE as described above (section 2.4) were transferred to nitrocellulose membranes in a SemiPhor transfer system (Hoefer Scientific Instruments). Membranes were immersed in Towbin Solution (25mM Tris-HCl, pH 8.3, with 192mM glycine, 20% methanol, and 1.3mM SDS) for 20 min, and transferred using the same buffer for 1h, at 0.8mA/cm2. Membranes were blocked overnight (at 4°C) in 10% non-fat milk in TBST (50mM Tris-HCl, pH 7.5, with 150mM NaCl and 0.05% Tween 20), and incubated with a monoclonal antibody against HSP70 (clone BRM-22, Sigma Chemical Co.) at room temperature at a dilution of 1:5,000 for 1h. After washing thrice (10 min each) in TBST, membranes were incubated with anti-mouse peroxidase-linked secondary antibodies (ECL, Amersham, UK) diluted at 1:1,000, for 1h at room temperature. Labelling was visualized via ECL (Amersham) using Kodak X-Omat K film.

RESULTS

PGA1 inhibits MAYV replication in Hep-2 cells

To evaluate the effect of PGA1 on MAYV replication in Hep-2 cells, virus proliferation in these cells was analyzed under different concentrations of PGA1. Following the adsorption period with an MOI of 1, cells were treated with PGA1 at specific times, and culture supernatants were collected for titration.

At 48 hours post-infection (hpi), PGA1 inhibited MAYV replication by 50% and 83% at 0.1 and 1µg/mL PGA1, respectively (Figure 1A). Effect of higher concentrations of PGA1 on MAYV replication at was examined (Figure 1B). While 1µg/mL PGA1 inhibited MAYV production by 50% at, higher concentrations inhibited viral production by 82 (2µg/mL), 90 (4µg/mL) and 95% (6µg /mL) at (Figure 1B). Viability tests using neutral red showed that PGA1 is not cytotoxic under tested conditions (data not shown).

Effect of PGA1 on MAYV replication at different times post-infection was evaluated (Figure 1C). The inhibition of MAYV replication was high (~90%) when PGA1 (5µg/mL) was added soon (within 2h) after adsorption (Figure 1C). When PGA1 was added post-infection, its inhibitory potential decreased (Figure 1C).

FIGURE 1:
Effect of PGA1on MAYV replication. Hep-2 cells were infected with MAYV (MOI=1) and treated with PGA1 at different concentrations and times post-infection. A. Lower PGA1 concentrations (0.1, 0.5 and 1µg/mL) were added after the adsorption period, and the viral titer in the supernatant of infected cells was evaluated at 6, 20, 24 and 48 hpi. B. Effect of higher concentrations of PGA1 (1 to 6µg/mL, for 24h) on viral replication in infected cells, relative to the controls - infected cells kept untreated. C. Inhibition of viral replication after treatment with 5µg/mL PGA1 added at different times post-infection. PFU/mL: Plaque-forming unit; PGA 1 : Prostaglandin A1; MAYV: Mayaro virus; MOI: multiplicity of infection; hpi: hours post-infection.

Effect of PGA1 treatment before infection initiation was examined (Table 1). Cells were infected and treated with PGA1 (5µg/mL) 12h before infection initiation, and at 0, 1, and 3 hpi (Table 1). For pre-treatment with PGA1, cells were washed with PBS prior to MAYV infection, which was performed for 24 hpi, and supernatants were processed for virus titration. PGA1 treatment at 1 and 3 hpi yielded similar inhibition values, confirming our earlier results (Figure 1C), but PGA1 treatment 12h before infection did not inhibit MAYV production (Table 1).

TABLE 1:
Effect of PGA1 treatment on MAYV production by Hep-2 cells.

To analyze effects of PGA1 on MAYV replication, Hep-2 cells were infected (MOI = 5), treated with 5µg/mL PGA1 for 24h, and examined via transmission electron microscopy. The treatment did not alter the overall ultrastructure of uninfected Hep-2 cells (Figure 2A and Figure 2B). Untreated, infected cells exhibited many mature viral particles adhering to the cell membrane at the extracellular domain, and others budding from the plasma membrane (Figure 2C, Figure 2D and Figure 2E). A marked reduction in the number of virus particles was observed in cells treated with 5µg/mL PGA1. No viral precursors or modified vacuoles typical of viral morphogenesis inhibition were seen in the cytoplasm of treated cells, which had much fewer virus-like particles budding from the plasma membrane than untreated cells (Figure 2F and Figure 2G).

FIGURE 2:
Transmission electron microscopy of Hep-2 cells infected with MAYV and treated with PGA1. Cells were treated with PGA1 (5µg/mL) for 24h concomitantly with infection with MAYV. A and B General views of the periphery from uninfected cells kept untreated (A) or treated with 5µg/mL PGA1 (B) showing no clear structural changes after treatment. (C, D and E) Untreated cultures infected with MAYV (MOI=5) for 24h had a large number of viral particles being released to the intercellular region (C) after budding from the plasma membrane D. (arrows). F and G. Infected cells treated with PGA1 for 24h, showing a drastic reduction in the release of viral particles (F, arrows), as well as the absence of nucleocapsids from the host cell cytoplasm (G). n: nucleus. Scale bars: 100nm (A-E); 200nm (F-G). Hep-2: Human cervix carcinoma cells; MAYV:Mayaro virus; PGA 1 : Prostaglandin A1; MOI: multiplicity of infection.

Effect of PGA1 on cellular and viral protein synthesis

In addition to the effect of PGA1 on virus production, we analyzed the effect of PGA1 on viral and cellular protein production, via 3535. Pinheiro FP, Freitas RB, Travassos JF, Da Rosa YBG, MelloWA, LeDuc JW. An outbreak of Mayaro virus disease in Belterra, Brazil. I. Clinical and virological findings. Am J Trop Med Hyg .1981;30(3):674-81.S-methionine cell labeling (for 60 min, 24 and 48 hpi), followed by SDS-PAGE and autoradiography analysis. As PG induces the expression of HSPs3030. de Marco A, Carattoli A, Rozera C, Fortini D, Giorgi C, Belardo G, et al. Induction of the heat-shock response by antiviral prostaglandins in human cells infected with human immunodeficiency virus type 1. Eur J Biochem. 1998;256(2):334-41.,3131. Sugiyama R, Nishitsuji H, Furukawa A, et al. Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G. J Biol Chem. 2013;286(12):10051-7.,3333. Hamel L, Kenney M, Jayyosi Z, Ardati A, Clark K, Spada A, et al. Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells. Cell Stress Chaperones. 2000;5(2):121-31., we analysed whether HSP70 expression was induced after PGA1 treatment. The controls consisted of uninfected and untreated cells subjected to heat-shock treatment (incubation at 42°C, for 1h) before 3535. Pinheiro FP, Freitas RB, Travassos JF, Da Rosa YBG, MelloWA, LeDuc JW. An outbreak of Mayaro virus disease in Belterra, Brazil. I. Clinical and virological findings. Am J Trop Med Hyg .1981;30(3):674-81.S-methionine labelling.

PGA1 inhibited viral protein synthesis drastically at 48 hpi (p110, p62, E1/E2 and C), but could not reverse the inhibition of cellular protein synthesis induced by MAYV infection (Figure 3A). Densitometry analysis suggested that PGA1 inhibited the production of MAYV proteins by ~15-30% and ~50%, at 24 and 48 hpi, respectively (Figure 3B).

Pronounced induction of HSP70 expression was observed when cells were subjected to heat shock (Figure 3A). To evaluate the effect of PGA1 on HSP70 expression more precisely, western blot labelled with an anti-HSP monoclonal antibody was used. Heat-shock treatment increased HSP70 expression ~241 fold, relative to the untreated control, while 1 and 5µg/mL of PGA1 increased HSP70 levels by 52% and 130%, respectively (Table 2).

FIGURE 3:
Cellular and viral protein synthesis during PGA1treatment of cells infected with MAYV. A. Cells were infected (MOI=1) and treated with PGA1 (5µg/mL) for 24 and 48 hpi, incubated with 3535. Pinheiro FP, Freitas RB, Travassos JF, Da Rosa YBG, MelloWA, LeDuc JW. An outbreak of Mayaro virus disease in Belterra, Brazil. I. Clinical and virological findings. Am J Trop Med Hyg .1981;30(3):674-81.S-methionine (30µCi/mL) for 60 min and prepared for SDS-PAGE analysis. Samples are labeled as follows: C, control (mock infected, untreated) cells; V, MAYV infected cells; PG, uninfected cells treated with PGA1; V+PG, infected cells treated with PGA1; and T, uninfected and untreated cells subjected to heat shock (42°C, 1h), as a control for HSP70 induction. The expected positions of viral proteins (p110, p62, E1/E2, C) are indicated on the right. B. Densitometric analysis of MAYV protein levels detected a slight inhibition (15%) in the synthesis of viral structural proteins at 24hpi, and this effect increased to 45-55% at 48 hpi. HSP: heat shock protein; PGA 1 : Prostaglandin A1; MAYV: Mayaro virus; MOI: multiplicity of infection; Ci/mL: Curie; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; hpi: hours post-infection.

TABLE 2:
Densitometric analysis of HSP70 protein levels by western blot, after treatment of MAYV-infected cells with 1 and 5µg/mL of PGA1 for 24h.

DISCUSSION

Bird migration, and human migration and travel, combined with high mutation rates of RNA polymerases are some factors that may increase the risk of an MAYV outbreak in the Americas33. Esposito DLA, Fonseca BALD. Will Mayaro virus be responsible for the next outbreak of an arthropod-borne virus in Brazil? Braz J Infect Dis. 2017;21(5)540-4.,3131. Sugiyama R, Nishitsuji H, Furukawa A, et al. Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G. J Biol Chem. 2013;286(12):10051-7.,3434. Peña-García VH, McCracken MK, Christofferson RC. Examining the potential for South American arboviruses to spread beyond the New World. Curr Clin Microbiol Rep. 2017;4(4):208-17.. Neither vaccines nor effective treatments are available against human MAYV infection yet. Considering the epidemiological potential of MAYV, and Chikungunya-like symptoms that hinder differential diagnoses between Chikungunya and Mayaro3535. Pinheiro FP, Freitas RB, Travassos JF, Da Rosa YBG, MelloWA, LeDuc JW. An outbreak of Mayaro virus disease in Belterra, Brazil. I. Clinical and virological findings. Am J Trop Med Hyg .1981;30(3):674-81., it is important to identify treatments capable of inhibiting MAYV replication.

We demonstrated that PGA1 may inhibit MAYV production in Hep-2 cells in a dose-dependent manner, with 2µg/ml PGA1 inhibiting ~80% of virus production. PGA1 inhibited MAYV replication at lower concentrations than those required for Sendai virus inhibition2424. Santoro MG, Amici C, Elia G, Benedetto A, Garaci E. Inhibition of virus protein glycosilation as the mechanism of the antiviral action of prostaglandin A in Sendai virus-infected cells. J Gen Virol. 1989;70(Pt 4):789-800.. It is noteworthy that PGA1 treatment had no detectable effect on cellular viability even at higher doses (10µg/ml), during the treatment times analyzed, (not shown).

Santoro et al.2424. Santoro MG, Amici C, Elia G, Benedetto A, Garaci E. Inhibition of virus protein glycosilation as the mechanism of the antiviral action of prostaglandin A in Sendai virus-infected cells. J Gen Virol. 1989;70(Pt 4):789-800., reported that PGs do not significantly affect early stages of viral infection, namely adsorption, penetration and uncoating2424. Santoro MG, Amici C, Elia G, Benedetto A, Garaci E. Inhibition of virus protein glycosilation as the mechanism of the antiviral action of prostaglandin A in Sendai virus-infected cells. J Gen Virol. 1989;70(Pt 4):789-800.. However, PGA1 treatment at 4 hpi, reduced antiviral potential compared to PGA1 treatment at earlier stages of infection (1-3 hpi). Similar results have been reported regarding PGA1 inhibition of HIV replication2727. Ankel H, Turriziani O, Antonelli G. Prostaglandin A inhibits replication of human imunodeficiency virus during acute infection. J Gen Virol . 1991;72(Pt 11):2797-800., corroborating the hypothesis that initial stages of infection may be important targets for PG inhibition of MAYV replication.

Some antiviral molecules, including interferon3636. Sen GC, Ransohoff RM. Interferon-induced antiviral actions and their regulation. Adv Virus Res. 1993;42:57-102., may inhibit viral replication even when cells are treated before infection. However, pre-treatment of cells with PGA1 had no protective effect against MAYV infection.

We showed that some antiviral substances (e.g., interferon, monensin, and weak bases) induce remarkable defects in viral particle morphogenesis3737. Rebello MCS, Fonseca MEF, Marinho JO, Rebello MA. Interferon action on Mayaro virus replication. Acta Virol. 1993;37(4):223-31.

38. De Campos RM, Ferreira DF, Da Veiga VF, Rebello MA, Rebello MC. Effect of monensin on Mayaro virus replication in monkey kidney and Aedes albopictus cells. Acta Virol . 2003;47(2):113-9.
-3939. Ferreira DF, Santo MPE, Rebello MA, Rebello MCS. Weak bases affect late stages of Mayaro virus replication cycle in vertebrate cells. J Med Microbiol. 2000;49(4):313-8.. Superti et al.2828. Superti F, Amici C, Tinari A, Donelli G, Santoro MG. Inhibition of Rotavirus replication by prostaglandin A: evidence for a block of virus maturation. J Infect Dis. 1998;178(2):564-8. observed that in monkey kidney cells (MA-104) infected with Rotavirus, and treated with PGA1, normal cytoplasmic inclusions and particles budding from the endoplasmic reticulum were present, but viral maturation was impaired. Our analysis suggests, however, that PGA1 does not disrupt MAYV morphogenesis in Hep-2 cells, as evidenced by the lack of nucleocapsid accumulations in the cytoplasm following PGA1 treatment, and the drastic reduction in viral particles in intercellular spaces. These results indicate that the severe decrease in the viral titer after treatment, may, at least in part, be due to structural protein synthesis inhibition by PGA1.

Bader and Ankel2626. Bader T, Ankel H. Inhibition of primary transcription of Vesicular Stomatitis virus by prostaglandin A1. J Gen Virol . 1990;71(Pt 12):2823-32. showed that inhibition of viral RNA polymerase activity may be a potential mechanism underlying VSV replication inhibition by PGA1. This may explain the decrease in viral messenger RNA synthesis observed in cells treated with PGA14040. Parker J, Ahrens PB, Ankel H. Antiviral effect of cyclopentenone prostaglandins on vesicular stomatitis virus replication. Antiviral Res. 1995;26(1):83-96.. Inhibition of primary transcript production may potentially explain the antiviral effect of PGA1. However, RNA polymerase inhibition may not fully clarify other patterns of DNA and RNA virus replication inhibition by PGs, which may be mechanistically more complex.

Western blot analysis using anti-HSP70 monoclonal antibodies showed that PGA1 treatment increases HSP70 expression. These results confirm those of other studies showing that viral replication inhibition by cyclopentane PGs (PGA1 and PGJ2) is associated with the ability of these molecules to trigger HSP70 synthesis in a large variety of animal cells4141. Santoro MG. Heat shock proteins and virus replication: HSP70s as mediators of the antiviral effects of prostaglandins. Experientia. 1994;50(11-12):1039-47.,4242. Santoro MG. Viral infection. In: Feige U, Morimoto RI, Yahara I, Polla B, editos. Stress-inducible cellular responses. Basel, Switzerland: Birkhäuser Verlag; 1996. p. 336-57.. This phenomenon is mediated by the heat shock factor (HSF), a transcriptional activator for HSPs4343. Holbrook NJ, Carlson SG, Choi AM, Fargnoli J. Induction of HSP70 gene expression by the antiproliferative prostaglandin PGA2: a growth-dependent response mediated by activation of heat shock transcription factor. Mol Cell Biol. 1992;12(4):1528-34.,4444. Santoro MG. Heat shock factors and the control of the stress response. Biochem Pharmacol. 2000;59(1):55-63.. Several studies demonstrated that HSP70 is required for viral replication inhibition by PGA1. HSP70 inhibition decreased antiviral activity of PGA1 against MAYV in Vero cells4545. Burlandy FM, Rebello MA. Inhibition of Mayaro virus replication by prostaglandin A(1) in Vero cells. Intervirology. 2001;44(6):344-9.. The inhibitory effect of PGA1 against the Sindbis virus was abolished by HSP70 induction4646. Mastromarino P, Conti C, Petruzziello R, De Marco A, Pica F, Santoro MG. Inhibition of Sindbis vírus replication by cyclopentenone prostaglandins: a cell-mediated event associated with heat shock protein synthesis. Antiviral Res . 1993;20(3):209-22.. A variety of HSP70 inducers may block the replication of different virus types4747. de Meneses MDF, Rebello MA. Effect of prostaglandin A1, arsenite and aspirin on stress proteins response in mosquito cells. Z Naturforsch C. 2001;56(3,4):298-302.,4848. Carvalho MGC, Rebello MA, Mezencio JMS. Effect of high temperature on Aedes albopictus cells infected with Mayaro virus. Braz J Med Biol Res . 1987;20(6):857-60..

Due to different viral replication inhibition strategies of PGs, it is difficult to establish a general antiviral mechanism for HSP activation associated with PG treatment. HSPs may control viral replication by interacting with viral proteins, as observed in VSV and enterovirus, among others4949. Pica F, Rossi A, Santirocco N, Palamara A, Garaci E, Santoro MG. Effect of combined alpha IFN and prostaglandin A1 treatment on vesicular stomatitis virus replication and heat shock protein synthesis in epithelial cells. Antiviral Res . 1996;29(2-3):187-98.,5050. Macejak DG, Sarnow P. Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells. J Virol. 1992;66(3):1520-7.. Alternatively, HSPs may downregulate viral protein synthesis, as HSP70 synthesis triggered by PGA1 treatment during initial stages of infection, has been associated with viral protein synthesis inhibition and a reversal of cellular protein synthesis inhibition induced by the virus4141. Santoro MG. Heat shock proteins and virus replication: HSP70s as mediators of the antiviral effects of prostaglandins. Experientia. 1994;50(11-12):1039-47.. We observed that PGA1 inhibited MAYV protein synthesis by 50% at 48 hpi. However, the profile of cellular proteins was not altered by PGA1, which did not reverse virus-induced inhibition of cellular protein synthesis, after 48h. While PGA1 inhibited viral protein synthesis by 15% at 24 hpi, viral replication was inhibited by 95%, indicating that viral mRNA translation is not the main target of the compound, which corroborates previous data on the effects of PGA and PGJ on poliovirus infection5151. Conti G, Mastromarino P, Tomao P, De Marco A, Pica F, Santoro MG. Inhibition of poliovirus replication by prostaglandins A and J in human cells. Antimicrob Agents Chemother. 1996;40(2):367-72..

Although 3535. Pinheiro FP, Freitas RB, Travassos JF, Da Rosa YBG, MelloWA, LeDuc JW. An outbreak of Mayaro virus disease in Belterra, Brazil. I. Clinical and virological findings. Am J Trop Med Hyg .1981;30(3):674-81.S-methionine labelling in non-infected cells after 24h of PGA1 treatment did not clearly indicate HSP70 induction, western blot analysis revealed a 130 fold increase in HSP70 levels following 5µg/mL PGA1 treatment. A low level of constitutive HSP70 expression was also detected in the control cells, and heat shock treatment induced higher HSP70 expression levels than those observed following PGA1 treatment. Possibly, relatively low levels of HSP70 synthesis induced by PGA1 in Hep-2 cells may be sufficient to inhibit MAYV replication. This result is different compared to that reported for numerous other cell lines, where PGA1 treatment did not induce HSP70 synthesis4242. Santoro MG. Viral infection. In: Feige U, Morimoto RI, Yahara I, Polla B, editos. Stress-inducible cellular responses. Basel, Switzerland: Birkhäuser Verlag; 1996. p. 336-57.,5252. Rossi A, Santoro MG. Induction of a 32-kDa stress protein by prostaglandin A1 in cultured murine cells. Ann N Y Acad Sci. 1994;744:326-9..

PGA1 and PGJ2 inhibit viral mRNA transcription, leading to the identification of nuclear factor kappa B (NF-κB) as a target for PGs3030. de Marco A, Carattoli A, Rozera C, Fortini D, Giorgi C, Belardo G, et al. Induction of the heat-shock response by antiviral prostaglandins in human cells infected with human immunodeficiency virus type 1. Eur J Biochem. 1998;256(2):334-41.. This factor remains inactive in eukaryotic cells unless activated by phosphorylation in response to different stimuli, including stimulation by cytokines as well as bacterial and fungal infections. NF-κB is then translocated to the nucleus where it induces the transcription of a variety of genes. In addition to being important immunological mediators, PGA1 and PGJ2 play a role in the inflammatory response by inhibiting NF-κB in human cells through blocking phosphorylation and degradation of the NF-κB inhibitor Iκ-B1919. Rossi A, Kapahi P, Natoli G, Takahashi T, Chen Y, Karin M, et al. Anti-inflamatory cyclopentenone prostaglandins are direct inhibitors of IκB kinase. Nature. 2000;403(6765):103-8.. Inhibition of NF-κB by PGA1 may explain some results of our present study, but further studies are needed to establish whether NF-κB modulation affects the observations made in our study.

Our results indicate that interaction between the Hep-2 cell line and MAYV may be different compared to a large number of cell lines examined in previous studies2323. Ishimaru D, Marcicano FG, Rebello MA. Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells. Braz J Med Biol Res. 1998;31(9):1119-23.,4545. Burlandy FM, Rebello MA. Inhibition of Mayaro virus replication by prostaglandin A(1) in Vero cells. Intervirology. 2001;44(6):344-9.,5353. Barbosa JA, Rebello MA. Prostaglandin A1 inhibits replication of mayaro virus in Aedes albopictus cell. Braz J Med Biol Res . 1995;28(1):27-30.. While we detected an increase in HSP70 expression in infected Hep-2 cells treated with PGA1 via western blot, HSP70-independent PGA1 modes of action may also be operating in this virus-cell system.

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  • Financial Support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Publication Dates

  • Publication in this collection
    Sep-Oct 2018

History

  • Received
    04 June 2018
  • Accepted
    07 Aug 2018
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