Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta

Abstract

Recently, the Tomato Spotted Wilt Virus (TSWV) Gn and Gc glycoproteins were shown to induce the formation of (pseudo-) circular and pleomorphic membrane structures upon transient expression in plant cells. Furthermore, when singly expressed, Gc retains in the ER, while Gn is able to further migrate to the Golgi. Upon co-expression, Gn rescues Gc and co-migrates to the Golgi complex. Here, we have studied the behavior of the glycoproteins in the presence of the viral nucleocapsid (N) protein and in vivo analyzed the occurrence of protein-protein interactions by fluorescence life time imaging microscopy (FLIM). The analysis demonstrated that N co-localizes and interacts with both glycoproteins, with a preference for Gn. Additionally, it is shown that N causes a dramatic change in the distribution of Gc within the ER, from reticular to punctate spots. The observations are discussed in the context of the virus particle formation during the infection process.

Cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein

“…To mediate the packaging of viral RNA, an enveloped virus that lacks a matrix protein instead requires a direct interaction between the intraviral domains of the envelope-embedded glycoproteins and the RNP. The most thoroughly characterized examples of this type of direct glycoprotein-RNP interaction have been described for the families Coronaviridae, Flaviviridae and Togaviridae (Lo et al, 1996;Narayanan et al, 2000;Sturman et al, 1980) and, while this is the first report of glycoprotein-RNP interaction for a virus in the genus Hantavirus, it has already been reported for other members of the family Bunyaviridae (Liu et al, 2008;Overby et al, 2007a;Ribeiro et al, 2009;Snippe et al, 2007). In addition, this interaction has recently been identified with the TULV Gn-CT and N protein using recombinant proteins (Wang et al, 2010).…”

“…The peptide-bound proteins were separated by SDS-PAGE (10 % gel) and detected by autoradiography (in vitro-translated N) or immunoblotting using rabbit antiserum to PUUV N protein (RNP). et al, 2007a;Ribeiro et al, 2009;Snippe et al, 2007). In addition, this interaction has recently been identified with the TULV Gn-CT and N protein using recombinant proteins (Wang et al, 2010).…”

“…It is common for enveloped viruses that the contacts between the RNP and the envelope et al, 2007b), and the CTs of both Gn and Gc of tospoviruses interact with the N protein (Ribeiro et al, 2009;Snippe et al, 2007). The nucleocapsid interaction of SFV is mediated by the cytoplasmic domain of the E2 glycoprotein (Metsikko & Garoff, 1990;Vaux et al, 1988).…”

“…By analogy to alphaviruses, the envelope glycoproteins of hantaviruses are likely to operate as a surrogate matrix that organizes the interactions between the envelope and the nucleocapsid. Within members of the family Bunyaviridae, the Gn-CT is required for binding to RNP in members of the genus Phlebovirus (Overby et al, 2007b), and the CTs of both Gn and Gc of tospoviruses interact with the N protein (Ribeiro et al, 2009;Snippe et al, 2007). The nucleocapsid interaction of SFV is mediated by the cytoplasmic domain of the E2 glycoprotein (Metsikko & Garoff, 1990;Vaux et al, 1988).…”

Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein ofTomato spotted wilt virus

“…These vRNP complexes perform genome RNA replication and viral mRNA transcription. Moreover, N protein has been shown to interact with Gn/Gc protein and it has therefore been suggested that the N protein plays a crucial role in viral encapsidation (Ribeiro et al, 2009). Overall, the N protein is an important factor in the TSWV life cycle.…”