Requirement of Cauliflower Mosaic Virus Open Reading Frame VI Product for Viral Gene Expression and Multiplication in Turnip Protoplasts

Abstract

Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post-transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame-shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co-transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co-transfection experiments with either pCa122 or pCaFS6. This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, IV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended on the co-expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication.

Dissection of Cauliflower Mosaic Virus Transactivator/Viroplasmin Reveals Distinct Essential Functions in Basic Virus Replication

“…Total omission of Tav resulted in a total block of CaMV replication (Fig. 2C and D, lanes 13), supporting our previous finding that Tav is essential for one or more steps of the CaMV replication cycle (19). TavD1, -D2, -D3, -D4, -D11, and -D12 supported CaMV DNA replication, although to dif- ferent levels: TavD4, nearly 100% of wild type; TavD2 and -D3, 50 to 70%; TavD12, 20%; TavD11, 5%; TavD1, 1% (Fig.…”

“…3A, lanes 6, 10, and 14). In contrast to our previous report (19), Gag accumulation was observed in the absence of Tav when Gag and Pol were provided from separate plasmids, although seemingly to much lower levels than those in the presence of the mutant TavD6 protein (cf. Fig.…”

“…Tav also interacts with a processed form of Gag and has been implicated in virion assembly (11). We have found that it stabilizes Gag and Pol and is essential for virus replication in single cells (19). Tav has also been shown to play a central role in pathogenesis in solanaceous plants (5,6,29,(35)(36)(37).…”

“…In contrast, Gag and Pol are probably essential for basic replication based on their similarity to retroviral Gag and Pol proteins. We have shown that Tav is also required for CaMV replication in transfected protoplasts (19).…”

“…Since we have shown that Tav can stabilize Gag and Pol in transfected cells (19), we examined if CaMV DNA replication could occur in cells cotransfected with pE4Pin and Gag and Pol expression plasmids but not with the Tav expression plasmid. The level of CaMV DNA replication when Gag and Pol were provided from monocistronic plasmids (in the absence of Tav) was about 10,000 times lower than that when Gag and Pol were expressed from the polycistronic plasmid in the presence of Tav (Fig.…”