Membrane association of a nonconserved viral protein confers virus ability to extend its host range

Abstract

Citrus tristeza virus (CTV), the largest and most complex member of the family Closteroviridae, encodes a unique protein, p33, which shows no homology with other known proteins, however, plays an important role in virus pathogenesis. In this study, we examined some of the characteristics of p33. We show that p33 is a membrane-associated protein that is inserted into the membrane via a transmembrane helix formed by hydrophobic amino acid residues at the C-terminal end of the protein. Removal of this transmembrane domain (TMD) dramatically altered the intracellular localization of p33. Moreover, the TMD alone was sufficient to confer membrane localization of an unrelated protein. Finally, a CTV variant that produced a truncated p33 lacking the TMD was unable to infect sour orange, one of the selected virus hosts, which infection requires p33, suggesting that membrane association of p33 is important for the ability of CTV to extend its host range.

The conundrum of a unique protein encoded by citrus tristeza virus that is dispensable for infection of most hosts yet shows characteristics of a viral movement protein

“…2A, white arrows). Similar distribution of the GFP-tagged p33 was observed also under the conditions when p33 was expressed from the native promoter upon virus infection (Kang et al, 2015). Here we extended our microscopy observations to examine the details of the intracellular localization of p33 in order to identify association of the protein with particular cellular structures.…”

“…Agrobacterium tumefaciens-mediated delivery of a cassette encoding the fusion protein into leaves of N. benthamiana plants allowed observation of the distribution of GFP-tagged p33 in cells of the infiltrated leaves. In our recent work, we showed that the GFP:p33 fusion protein accumulated at the cytoplasmic membrane and also formed vesicular bodies distributed throughout the cytoplasm but not in the nucleus (see Kang et al (2015) and Fig. 2A, white arrows).…”

“…In our recent study we showed that p33 is a membraneassociated protein that is anchored to the membrane via a C-terminal transmembrane domain (Kang et al, 2015). We also demonstrated that disruption of the membrane localization of p33 affected virus ability to infect specific hosts (Kang et al, 2015).…”

“…To analyze the intracellular localization of p33, we used a construct in which the p33 gene was fused to the green fluorescent protein (GFP) gene and placed under the 35 S promoter in a binary vector (pGFP:p33; Kang et al, 2015). Agrobacterium tumefaciens-mediated delivery of a cassette encoding the fusion protein into leaves of N. benthamiana plants allowed observation of the distribution of GFP-tagged p33 in cells of the infiltrated leaves.…”

“…We also demonstrated that disruption of the membrane localization of p33 affected virus ability to infect specific hosts (Kang et al, 2015). Here we examined the subcellular distribution of the p33 protein in more detail in order to obtain additional information that would shed some light on its function.…”