Guest Writer: Adam C. Winterhalter

The potyvirus group (named for its prototypical member, potato virus Y (PVY)) is the largest of the 34 plant virus groups and families currently recognised (Ward & Shukla, 1991). This group contains at least 180 definitive and possible members (30% of all known plant viruses) which cause significant losses in agricultural, pastural, horticultural and ornamental crops (Ward & Shukla, 1991).

Potyviruses are quite similar, in terms of their genomic structure and strategy of expression, to the plant bipartite como- and nepoviruses, and to the animal picornaviruses. In addition, the genome of these differing species contains a region of conserved gene order which encodes non-structural proteins involved in RNA replication. For these reasons it has been proposed that the como-, nepo- and potyviruses may be arranged in a supergroup of picorna-like plant viruses (Goldbach, 1986, 1987; Goldbach, et al., 1990).

Potyvirus virions are non-enveloped (Langenberg & Zhang, 1997) filamentous particles, 680 to 900nm long and 11 to 15nm wide (Dougherty & Carrington, 1988; Riechmann, et al., 1992). The definitive morphological structure is composed of approximately 2000 copies of capsid protein (CP) (Martin & Gelie, 1997) which encapsidates a single stranded, positive sense RNA genome approximately 10kb in length which has a 5' terminal linked protein (VPg) (Hari, 1981; Siaw et al., 1985; Riechmann et al., 1989; Murphy et al., 1990), and a 3' poly-A tail (Hari, et al., 1979; Takahashi, et al., 1997). The positive sense genome can act directly as a messenger RNA, with the 5' non-coding region functioning as an enhancer of translation (Carrington & Freed, 1990). Despite the genetic relationship among potyviruses, picornaviruses and comoviruses, the mechanism of internal ribosomal entry which has been demonstrated or suggested for these last two groups of viruses (Thomas, et al., 1991) has not been found to operate for translation of potyvirus RNA. More likely, translation is initiated by recognition of an internal AUG codon by a leaky scanning mechanism (Riechmann, et al., 1991).

The RNA genome contains one long open reading frame (ORF) expressed as a 350kDa polyprotein precursor (Riechmann, et al., 1992; Riechmann et al., 1995). This is proteolytically processed by viral and host proteases (Langenberg & Zhang, 1997) into seven smaller proteins denoted as P1, helper component (HC), P3, cylindrical inclusion (CI), nuclear inclusion A (NIa), nuclear inclusion B (NIb), capsid protein (CP), aswell as two small putative proteins known as 6K1 and 6K2 (Riechmann, et al., 1992).

The viral genome encodes a large polyprotein that is processed by three virus-encoded proteinases to yield the mature products. Two proteinases, P1 and the helper component proteinase (HC-Pro), catalyse only autoproteolytic reactions at their respective C termini (Carrington, et al., 1989; Verchot, et al., 1991). The remaining cleavage reactions are catalysed by either trans-proteolytic or autoproteolytic mechanisms by the small nuclear inclusion protein (NIa-Pro), an evolutionary homologue of the picornavirus 3C proteinase (Carrington and Dougherty, 1987a; Carrington and Dougherty, 1987b).

The processing and function of these many proteins is still controversial, but it is believed that they may be multifunctional (Riechmann et al., 1992; Verchot & Carrington, 1995; Mahajan, et al., 1996), and Table 1 outlines the possible functions suggested for these proteins.

PROTEIN	POSSIBLE FUNCTION(S)
P1	Proteinase;
	Cell-to-cell movement (speculation).
HC-Pro	Aphid mediated transmission;
	Proteinase;
	Cell-to-cell movement (speculation).
P3	Unknown (possible role in replication)
CI	Genome replication (RNA helicase);
	Membrane attachment;
	Nucleic acid stimulated ATPase activity;
	Cell-to-cell movement (speculation).
CP	RNA encapsidation;
	Involved in vector transmission;
	Cell-to-cell movement.
NIa-VPg	Genome replication (Primer for initiation of RNA synthesis).
NIa-Pro	Major Proteinase
NIb	Genome replication (RNA-dependent RNA polymerase [RdRp]).
6K1 & 6K2	Unknown, but possible roles in: - RNA replication;
	- Regulatory function inhibiting NIa nuclear translocation;
	- Membrane anchoring of replication machinery.

Figure 1 displays the organisation of the monopartite potyvirus genome, which is believed to be processed by the method depicted. Some potyvirus strains have a bipartite genome which contains identical proteins but are processed slightly different due to there being two strands of RNA and hence two polyproteins.

Figure 1: Monopartite potyviral genome displaying functional products after translation and cleavage. The size of each protein is noted above.

Within the potyvirus genome their are conserved and variable regions. The conserved regions incorporate the helper component proteinase (HC-Pro) and nuclear inclusion b (NIb), while the variable regions consist of P1, P3, and the coat protein (CP) (Aleman-Verdaguer et al., 1997). It has been reported that P3 displays low homology between species (Shukla et al., 1991) but despite the variation observed between P3 proteins of distinct potyviruses, few differences were noted within the P3 of YMV strains (Aleman-Verdaguer et al., 1997).

As the P3 protein was conserved between strains, then it must play an important role in the functioning of the virus. By identifying the localisation of P3 protein, a function may be able to be assigned. Therefore, Rodriguez-Cerezo, et al. (1993) raised antibodies against the 42KDa non-structural P3 protein of tobacco vein mottling virus (TVMV) and used them with immunogold labelling procedures to determine the subcellular location of P3 in infected Nicotiana tabacum cells. They reported findings of P3-specific gold label almost exclusively associated with the cylindrical inclusions typically formed in the cytoplasm of potyvirus infected cells. The P3 antibodies reacted with both the transverse (pinwheel-like) and longitudinal (bundle-like) orientation of these inclusion bodies.

However, Langenberg and Zhang (1997) conducted a similar experiment to Rodriguez-Cerezo, et al. (1993) but used tobacco etch virus (TEV) to determine the subcellular location of the P3 protein. Immunogold labelling with the antiserum showed labels associated with nucleoli, nuclei, or nuclear inclusions.

In newly infected cells, before formation of nuclear inclusions (NIs), nucleoli were the first to be labelled. Later in the infection process, after the formation of nuclear inclusions, all NIs were labelled with antibodies to the P3 protein, while nucleoli and CIs in the same cell were not specifically labelled. This suggests that the P3 protein migrated out of the nucleoli and into the nuclear matrix where it became part of nuclear inclusions as the infection progressed, demonstrating that TEV P3 protein is a third non-structural viral protein of nuclear inclusions (ie. NIa-VPg, NIb & P3).

The reason for P3 to be directed to the nucleus may be to temporarily inhibit host gene expression, much like the role of the poliovirus 2A protein which shows similarities to P3 (Dougherty & Carrington, 1988; Wang & Maule, 1995).

The discrepancies of P3 localisation between the two papers can not be directly explained, except that they do have one common denominator, that being P3 was always found associated with proteins involved in viral RNA replication (ie. CI as a helicase, NIb as an RdRp, and VPg as a genome linked protein). Therefore, this evidence suggests that the P3 protein may be involved in the replication of potyviral RNA. This hypothesis is also strongly supported by the discovery that mutants of TVMV with altered P3 coding regions prevent the detection of progeny viral RNA in infected protoplasts or plant cells, thus suggesting the involvement of this protein in replication of potyviral RNA (Rodriguez-Cerezo, et al., 1993).

However, there have been other functions suggested for P3, being:
   (i) involvement in RNA replication, because of its association with CIs of TVMV (Rodriguez-Cerezo, et al., 1993).
   (ii) involvement in early stages of viral replication, due to presence of P3 in nucleoli and nuclear inclusions (Langenberg & Zhang, 1997).
  (iii) a movement protein (Dougherty & Semler, 1993).
  (iv) a proteinase or protease cofactor (Dougherty & Carrington, 1988; Riechmann, et al., 1992).

Upon inspection of the primary structure of the P3 protein of TEV, it is revealed as being a hydrophobic protein (Kyte and Doolittle, 1982; Engelman, et al., 1986) which would suggest a possible localisation in membranes. Experiments by Rodriguez-Cerezo & Shaw (1991) found the presence of TVMV P3 protein in the membrane fraction of virus-infected cells which completely contradicts the suggested role of viral replication, and may elucidate a secondary function of P3.

Obviously, very little is known of the exact function of the P3 protein and some uncertainty also exists as to its size. The size of the P3 protein has been reported to range from about 38 to 50 kDa (Dougherty & Carrington, 1988; Robaglia, et al., 1989; Goldbach, 1992) but this may be associated with incomplete processing of the 6K protein.

It is also interesting to note that expression of the TEV-P3 protein gene in many lines of transformed tobacco plants resulted in plants being highly resistant to infection by TEV (Langenberg & Zhang, 1997). The reason behind this is unknown.

Therefore, P3 is most probably involved in viral replication but its exact function is still unknown. I'm sure it will only be a matter of time before the function of P3 is elucidated, and then this may provide insight into the generation of transgenic plants that display resistance to potyviral infection.

REFERENCES:

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