A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

Abstract

Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels-the cellular and the whole-organism levels-by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondaryinfection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems.

The Multiplicity of Cellular Infection Changes Depending on the Route of Cell Infection in a Plant Virus

“…Our MOI estimate during cell-to-cell movement of TuMV (MOI of Ϸ30 genomes/cell) is much higher than those previously reported for any other virus (for a review, see reference 45), in particular for other plant RNA viruses, such as Tobacco mosaic virus (TMV) (29), Soilborne wheat mosaic virus (SBWMV) (30), Tobacco etch virus (TEV) (46), and Citrus tristeza virus (CTV) (32). The MOI ranges between 5 and 6 for SBWMV and between 1 and 2 for TMV, TEV, and CTV.…”

“…Unfortunately, thus far, formally connecting these coinfection patterns to distinct MOIs is difficult due to the paucity of data. The MOI has been quantified in only a few multicellular host-virus models: an insect virus (27), an arbovirus in its mosquito vector (28), four plant viruses (each in a different host) (26,(29)(30)(31)(32), and HIV (33)(34)(35). In all cases the estimated values ranged from one to a few genome units per cell, suggesting that the MOI generally is low.…”

RNA Silencing May Play a Role in but Is Not the Only Determinant of the Multiplicity of Infection

“…The corollary is that the MOI would be controlled at least in part by virus functions, and evidence derived mostly from the study of bacteriophages (31,(33)(34)(35) indicates that this is the case. Although it has been reported that the CTV-encoded protein p33 was required for superinfection exclusion at the plant and tissue level, it did not seem to have a role in MOI determination (23,28), and for plant viruses, there is no evidence of a role of viral functions in the control of the MOI.…”

“…Bergua et al (23) estimated the MOI in phloem-associated cells of young citrus trees that were infected with a variant of Citrus tristeza virus (CTV) (genus Closterovirus) and later superinfected with a second variant, with each CTV variant being tagged with two fluorescent proteins and both lacking the p33 protein. The MOI value was also low, ranging from 1.06 to 1.07 in different plant sections (23). Finally, Gutiérrez et al (24) obtained different estimates of the MOI at two phases of the systemic infection of a single systemically infected leaf of turnip plants by Turnip mosaic virus (TuMV).…”

“…The exclusion of Vesicular stomatitis virus (VSV) (genus Vesiculovirus) defective interfering particles by VSV in cell culture is at the level of virus penetration and requires the expression of viral genes and a VSV transmembrane glycoprotein (35). Superinfection exclusion of CTV at the whole-organism level, but not at the cellular level, requires the CTV protein p33, which is not essential for CTV infection in most host plants (23,28). Thus, the information available on virus-encoded functions regulating cell infection by different viruses or genotypes is quite limited.…”