Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

Abstract

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3-untranslated regions (UTRs) of both RNAs deleted (R1⌬/2⌬ construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1⌬/2⌬ construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1⌬/2 (3-UTR of RNA 1 deleted), R1/2⌬ (3-UTR of RNA 2 deleted), or R1⌬/2⌬ plus R3. This demonstrates that the 3-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3-termini of AMV RNAs is not required for assembly of virus particles.RNA-dependent RNA polymerase (RdRp) proteins of many plus-strand RNA viruses have been expressed in heterologous systems, including Escherichia coli (15, 23-25, 31, 35, 42, 61), Saccharomyces cerevisiae (17, 36), insect cells (4,14,30,43,63), and mammalian cells (26). The availability of in vitro active RdRp has greatly facilitated research into the mechanisms of plus-strand virus replication. Mutant RNA polymerases can be expressed despite their inability to support viral replication. In this way, insight can be gained into the roles of the various domains of viral replicase proteins.Alfalfa mosaic virus (AMV) (for reviews, see references 6 and 18) is a tripartite plus-strand RNA virus belonging to the family Bromoviridae (Fig. 1A). The AMV replication complex consists of two viral proteins, P1 and P2, and possibly host proteins. P1, encoded by RNA 1, has homologies to known methyltransferase domains in its N-terminal half and homologies to helicase domains in its C-terminal half (12, 21). P2, encoded by RNA 2, contains the RNA polymerase domain characterized by the GDD motif (for a review, see reference 33). AMV RNA 3 codes for the viral movement protein (P3) and the coat protein (CP), which is translated from a subgenomic mRNA 4. All three genomic RNAs of AMV contain 5Ј-and 3Ј-untranslated regions (UTRs), which are highly structured and presumed to contain important cis-acting regulatory elements for viral replication and translation (32,52,54,55,58,59).The 3Ј-terminal 145 bases...

The 5′ Untranslated Region of Alfalfa Mosaic Virus RNA 1 Is Involved in Negative-Strand RNA Synthesis

“…Infiltration of N. benthamiana leaves with a mixture of Agrobacterium tumefaciens clones containing pMOGR12 and pMOGR3 results in accumulation of viral RNA and protein at the level of a wt AMV infection (31). pMOGR3 carries a 35S expression cassette containing cDNA 3 in its T region (31).…”

“…In the present study, the 5Ј UTR of RNA 1 (L1) was replaced with the 5Ј UTR of RNA 2 (L2) or RNA 3 (L3). The mutations were introduced into pBSR1, which contains a DNA copy of RNA 1 cloned between a cauliflower mosaic virus 35S promoter and the terminator of the nopaline synthase gene (31). To that end, sequences containing L2 or L3 downstream of a 35S promoter were transferred to pBSR1 from pCA27T-Nco or pCA35T-Nco using KpnI and NcoI.…”

“…35S expression cassettes encoding L2-R1 or L3-R1 were transferred from the respective pBSR1 derivatives to pMOGR2 using KpnI and SstI. pMOGR2 is a pMOG800 derivative carrying a 35S expression cassette containing cDNA 2, and pMOG800 is the transferred-DNA (T-DNA) vector used to express AMV RNAs in Nicotiana benthamiana by agroinfiltration (31). A derivative of pMOG800 carrying 35S expression cassettes of both RNAs 1 and 2 is called pMOGR12 (31).…”

“…pMOGR2 is a pMOG800 derivative carrying a 35S expression cassette containing cDNA 2, and pMOG800 is the transferred-DNA (T-DNA) vector used to express AMV RNAs in Nicotiana benthamiana by agroinfiltration (31). A derivative of pMOG800 carrying 35S expression cassettes of both RNAs 1 and 2 is called pMOGR12 (31). Infiltration of N. benthamiana leaves with a mixture of Agrobacterium tumefaciens clones containing pMOGR12 and pMOGR3 results in accumulation of viral RNA and protein at the level of a wt AMV infection (31).…”

In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

“…We do not think this effect is significant enough to support the model of the Gehrke lab, which states that low amounts of CP actually favor minus-strand synthesis (9). Previous experiments have shown that minusstrand RNA3 synthesis and plus-strand RNA4 synthesis can be perfectly carried out using a CP-free polymerase (5,10,28). Addition of CP to reactions with CP-free polymerase was found to block minus-strand synthesis.…”