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Journal of Virology, August 2009, p. 7487-7494, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00636-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Multiplicity of Infection of a Plant Virus Varies during Colonization of Its Eukaryotic Host{triangledown}

Pablo González-Jara,1 Aurora Fraile,1 Tomás Canto,2 and Fernando García-Arenal1*

Centro de Biotecnología y Genómica de Plantas (UPM-INIA) and E.T.S.I. Agrónomos, Universidad Politécnica de Madrid, Campus de Montegancedo, 28223 Pozuelo de Alarcón, Madrid, Spain,1 Centro de Investigaciones Biológicas (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain2

Received 27 March 2009/ Accepted 19 May 2009

The multiplicity of infection (MOI), i.e., the number of virus genomes that infect a cell, is a key parameter in virus evolution, as it determines processes such as genetic exchange among genomes, selection intensity on viral genes, epistatic interactions, and the evolution of multipartite viruses. In fact, the MOI level is equivalent to the virus ploidy during genome expression. Nevertheless, there are few experimental estimates of MOI, particularly for viruses with eukaryotic hosts. Here we estimate the MOI of Tobacco mosaic virus (TMV) in its systemic host, Nicotiana benthamiana. The progress of infection of two TMV genotypes, differently tagged with the green or red fluorescent proteins GFP and RFP, was monitored by determining the number of leaf cell protoplasts that showed GFP, RFP, or GFP and RFP fluorescence at different times postinoculation. This approach allowed the quantitative analysis of the kinetics of infection and estimation of the generation time and the number of infection cycles required for leaf colonization. MOI levels were estimated from the frequency of cells infected by only TMV-GFP or TMV-RFP. The MOI was high, but it changed during the infection process, decreasing from an initial level of about 6 to a final one of 1 to 2, with most infection cycles occurring at the higher MOI levels. The decreasing MOI can be explained by mechanisms limiting superinfection and/or by genotype competition within double-infected cells, which was shown to occur in coinfected tobacco protoplasts. To our knowledge, this is the first estimate of MOI during virus colonization of a eukaryotic host.

* Corresponding author. Mailing address: Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Universidad Politécnica de Madrid, Campus de Montegancedo, 28223 Pozuelo de Alarcón, Madrid, Spain. Phone: 34 91 3364550. Fax: 34 91 7157721. E-mail: fernando.garciaarenal{at}upm.es

{triangledown} Published ahead of print on 27 May 2009.

Journal of Virology, August 2009, p. 7487-7494, Vol. 83, No. 15
0022-538X/09/$08.00+0     doi:10.1128/JVI.00636-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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