INTERFERON SELECTIVELY INHIBITS THE SYNTHESIS OF MAYARO VIRUS GLYCOPROTEINS

Abstracts

We have previously observed that interferon (recIFN<FONT FACE="Symbol">a</FONT>2b) blocks the process of morphogenesis of Mayaro virus in TC7 cells (monkey kidney). In this work we show that IFNa inhibits preferentially virus glycoproteins and their precursors, and this effect is probably correlated to the alterations in the morphogenesis process previously observed.

Interferon <FONT FACE=Symbol>a</FONT>; Mayaro virus; glycoproteins; morphogenesis

Observamos anteriormente que o Interferon (IFN) recombinante <FONT FACE="Symbol">a</FONT>2b bloqueia o processo de morfogênese do vírus Mayaro em células TC7 (rim de macaco). Neste trabalho demonstramos que o IFN<FONT FACE="Symbol">a</FONT> inibe preferencialmente as glicoproteínas virais e seus precursores e que este efeito está, provavelmente, correlacionado com as alterações no processo de morfogênese previamente observadas.

Interferon <FONT FACE=Symbol>a</FONT>; vírus Mayaro; glicoproteínas; morfogênese

INTERFERON SELECTIVELY INHIBITS THE SYNTHESIS OF MAYARO VIRUS GLYCOPROTEINS

Davis F. Ferreira¹ and Maria Christina Soares Rebello^2*

¹Departamento de Virologia, Instituto de Microbiologia Professor Paulo de Góes, Rio de Janeiro, RJ, Brasil, ²Laboratório de Virologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, RJ, Brasil

Approved: July 23, 1998 SHORT COMMUNICATION

ABSTRACT

We have previously observed that interferon (recIFNa2b) blocks the process of morphogenesis of Mayaro virus in TC7 cells (monkey kidney). In this work we show that IFNa inhibits preferentially virus glycoproteins and their precursors, and this effect is probably correlated to the alterations in the morphogenesis process previously observed.

Key words: Interferon a, Mayaro virus, glycoproteins, morphogenesis

Mayaro virus is a member of the Alphavirus genus, Togaviridae family, and is perpetuated in nature by its ability to infect and replicate in vertebrate and invertebrate cells. The virus was firstly isolated in the Trinidad Island, in 1954, (1) and since then it has been isolated from humans and other mammals mainly at the colonization frontiers of the Amazon region (2). Clinically, Mayaro virus has been associated to symptoms similar to the Dengue fever, such as fever, myalgia or bone pain, frontal or retroorbital headache, rash, nausea, vomiting, etc. (1).

The 12-kb genome of the alphaviruses consists of an RNA molecule of positive polarity. The 5’ two-thirds of the genome code for the nonstructural proteins. The 3’ one-third of the genome codes for a polyprotein that will be processed and give rise to the structural proteins, consisting of a capsid protein (of about 30-KDa), a precursor of 62-Kda, which will undergo cleavage to produce two glycoproteins (E2 and E3), and finally another glycoprotein (E1). Usually only glycoproteins E1 and E2, of about 50-Kda each, are present at the final virus particle. (9).

Previous results in our laboratory showed that rec human alpha-2b interferon (IFN) is capable of inhibiting the production of Mayaro virus infectious particles from infected TC7 cells, inhibiting virus protein synthesis, recovering cellular protein synthesis and blocking Mayaro virus morphogenesis (7, 8). It was of our interest to study the mechanism of inhibition of virus morphogenesis by interferon by first analyzing the inhibition of each individual structural Mayaro virus proteins. TC7 cells were grown in Dulbecco modified Eagle’s medium containing 10% calf serum, and kept at 37°C. Mayaro virus was obtained from the American Type Culture Collection, Rockville, MD, USA. The virus stock was prepared from BHK-21 cells and stored at -70°C. Confluent monolayers were pre-treated with rec IFN a-2b (10³ UI/ml), from Shering-Plough S. A., and posteriously infected with Mayaro virus with a multiplicity of infection (moi) of 5. Incubation proceeded for 20 hours, when the cells were pulse-labeled (³⁵S methionine) and immuno precipitated with polyclonal antibody (a generous gift from Dr. Robert E. Shope, Yale Arbovirus Research Unit-Yale University, USA) and protein A purchased from Sigma Co. The samples were then analyzed by SDS-PAGE according to Laemmli (4). The resulting gel was exposed to a X-ray film Kodak X-Omat K, and the autorradiography analyzed (Fig. 1). The samples from the infected, not treated cells (Fig. 1, lane 1) allowed us to see the Mayaro virus proteins p34 (capsid protein), p50 (E2 protein), p52 (E1 protein), p62 (precursor protein), and p110 (another precursor protein). Comparing this protein pattern with the pattern observed for the infected-IFN pre-treated cells (lane 2) we evidence an overall inhibition of Mayaro virus proteins in the IFN pre-treated cells. However, such inhibition appears not to be homogeneous, affecting apparently more the glycoproteins and their precursors then the capsid protein. The autorradiogram was then submitted to a densitometric analysis using a Laser densitometer LKB, model 2202 (Fig. 2). The capsid protein is inhibited in 28%. The glycoproteins, however, showed a more expressive inhibition pattern. The glycoprotein precursors p110 and p62 were inhibited 84% and 77% respectively. The glycoprotein E1 (p52) was inhibited 99%, and the glycoprotein E2 (p50) was inhibited 79%.These data shows that the inhibition of virus structural protein synthesis is selective, that is, the glycoproteins and their precursors are most drastically inhibited by the IFN treatment than the capsid protein synthesis.

Figure 1 -
Analysis of the Mayaro virus protein synthesis in cells treated with IFN. TC7 cells were treated or not-treated with IFN 10³ IU/ml for 17 hours and infected with Mayaro virus (moi of 5). Cells were pulse-labeled with ³⁵S methionine at 20 hours post-infection, and processed for radio imuno precipitation. The proteins were analyzed by SDS-PAGE. The resulting autorradiography is shown. Lane 1, cells infected not-treated; lane 2, cells pre-treated and infected; lane 3, control not-infected cells.

Figure 2
- Densitometric analysis of the autorradiography from Fig. 1. The table shows the proteins visualized and their respective inhibition percentages comparing to infected not-treated cells.

This phenomenon could itself be highly responsible for the morphogenesis inhibition observed in our system. This selectivity of the glycoproteins by IFN was also observed for Herpes Simplex virus (HSV). Chatterjee et al. (1985) observed that IFN has little effect on the HSV capsid protein synthesis, but glycoproteins D and B had their synthesis drastically inhibited or retarded (3). Such report is very interesting, considering the fact that HSV are viruses in which structure and replication are completely different from the alphavirus.

Studies with Lb cells treated with IFN and infected with Vesicular Stomatitis Virus (VSV) (5) and HSV (6) demonstrated that the glycoprotein transport from trans golgi network to the plasma membrane is inhibited in these cells. They also found a raise in the intracellular pH (pHi) after the IFN treatment and suggested that one of the ways of action of IFN on the replication of certain viruses which affects mainly the glycoproteins would be by a pHi raise in such cells. The alkalinization of pHi could be in part responsible for the greater inhibition of the glycoproteins and their precursors, since their pathway through endoplasmic reticulum (ER) and golgi complex (GC) is extremely dependent of specific pH.

We are now investigating if the alkalinization of intracellular pH by IFN is an important factor on the effect of this agent on the replication of Mayaro virus, affecting the assembly as a consequence of the impaired processing of the virus glycoproteins. Studies are also in progress using immuno electron microscopy and pHi measurement in order to better elucidate the pH alterations by IFN in our system, relating to the glycoproteins pathway during processing.

RESUMO

Interferon a inibe seletivamente a síntese de glicoproteínas do vírus Mayaro.

Observamos anteriormente que o Interferon (IFN) recombinante a2b bloqueia o processo de morfogênese do vírus Mayaro em células TC7 (rim de macaco). Neste trabalho demonstramos que o IFNa inibe preferencialmente as glicoproteínas virais e seus precursores e que este efeito está, provavelmente, correlacionado com as alterações no processo de morfogênese previamente observadas.

Palavras-chave: Interferon a, vírus Mayaro, glicoproteínas, morfogênese.

REFERENCES

1. Anderson, D. R., Downs, W. G., Wattley, G. M., Alin, N. W., Reese, A. A. Mayaro virus: a new human disease agent II. Isolation from blood of patients in Trinidad. Am. J. Trop. Med. Hyg., 6: 1012, 1954.

2. Causey, O. R., Maroja, O. M. Mayaro virus: a new human disease agent III. Investigation of an epidemic of acute febrile illness on the River Guamá in Pará, Brazil and isolation of Mayaro virus as a causative agent. Am. J. Trop. Med. Hyg., 6:1017-1023, 1957.

3. Chatterjee, S., Hunter, E., Whittley, R. Effect of cloned human interferons on protein synthesis and morphogenesis of Herpes Simplex virus. J. Virol., 56: 419-425, 1985.

4. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London, 227: 680-685, 1970.

5. Maheshwari, R. K., Sidhu, D. B., Friedman, R. M. Primary amines enhanced the antiviral activity of interferon against a membrane virus: role of intracellular pH. J. Gen. Virol., 72: 2143-2152, 1991.

6. Maheshwari, R. K., Sidhu, G. S., Singh, A. K., Sivaram, S. S., Kinchington, P. R., Hay, J., Friedman, R. M. Defective transport of Herpes Simplex virus glycoprotein in interferon-treated cells: role of Intracellular pH. J. Interf. Res., 14: 319-324, 1994.

7. Rebello, M. C. S. Studies in Mayaro virus infected cells after interferon treatment. Anais da XXII Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular, p. 6, 1993.

8. Rebello, M. C. S., Fonseca, M. E. F., Marinho, J. O., Rebello, M. A. Interferon action on Mayaro virus replication. Acta Virol., 37: 223-231, 1993.

9. Strauss, J. H., Strauss, E. G. The Alphaviruses: Gene expression, replication, and evolution. Microbiol. Rev., 58: 491-562, 1994.

*

Corresponding author. Mailing address: Prof. Maria Christina Soares Rebello, Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Bloco G, Cidade Universitária, Ilha do Fundão, CEP 21949-900 Rio de Janeiro, RJ, Brasil. Fax: [phone].

Publication Dates

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

History

• Reviewed
  22 May 1998
• Received
  10 Sept 1997
• Accepted
  23 July 1998