In vivo packaging of brome mosaic virus RNA3, but not RNAs 1 and 2, is dependent on a cis-acting 3' tRNA-like structure

Abstract

The four encapsidated RNAs of brome mosaic virus (BMV; B1, B2, B3, and B4) contain a highly conserved 3' 200-nucleotide (nt) region encompassing the tRNA-like structure (TLS) which is required for packaging in vitro (Y. G. Choi, T. W. Dreher, and A. L. N. Rao, Proc. Natl. Acad. Sci. USA 99:655-660, 2002). To validate these observations in vivo, we performed packaging assays using Agrobacterium-mediated transient expression of RNAs and coat protein (CP) (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005). Coexpression of TLS-less constructs of B1 or B2 or B3 and CP mRNAs in Nicotiana benthamiana leaves resulted in packaging of TLS-less B1 and B2 but not B3, suggesting that packaging of B3 requires the TLS in cis. This conjecture was confirmed by the efficient packaging of a B3 chimera in which the viral TLS was replaced with a cellular tRNA(Tyr). When N. benthamiana leaves were infiltrated with a mixture of transformants containing wild-type B1 (wtB1) plus wtB2 plus a TLS-less B3 (wtB1+wtB2+TLS-lessB3), the 3' end of progeny B3 was restored by heterologous recombination with that of either B1 or B2. This intrinsic cis-requirement of TLS in promoting B3 packaging was further confirmed when a mixture containing agrotransformants of TLS-less B1+B2+B3 was supplemented with either wtB4 or a 3' 200-nt or 3' 336-nt untranslated region (UTR) of B3. Northern blot analysis followed by sequencing of B3 progeny revealed that replication of TLS-less B3, but not TLS-less B1 or B2, was fully restored due to recombination with TLS from transiently expressed wtB4 or the B3 3' UTR. Collectively, these observations suggested that the requirement of a cis-acting TLS is distinct for B3 compared with B1 or B2.

FIG. 1.

(A) Characteristics of T-DNA plasmids (agrotransformants) harboring BMV genomic RNAs used for Agrobacterium-mediated transient expression in plants. The 35S-B1, 35S-B2, and 35S-B3 constructs contain full-length cDNA copies of BMV genomic RNAs 1 (B1), 2 (B2), and 3 (B3), respectively (5). Filled arrows at the 5′ end represent the double 35S promoter (35S) whereas open boxes at the 3′ indicating RZ and T, indicate, respectively, the ribozyme sequence cassette derived from satellite tobacco ring spot virus and the 35S-polyadenylation terminator signals. The bent arrow represents the ribozyme cleavage site. The lengths of wt BMV RNAs and the number of nonviral nucleotides left after self-cleavage by ribozyme (shown in bracket) are indicated. (B) Characteristics of agrotransformants of BMV genomic RNAs lacking the 3′ TLS (ΔTLS). All other features are same those described for panel A. (C) Characteristic features of sgB4 agrotransformants of wild-type (35S-B4.1) and TLS-less (35S-B4.1/ΔTLS) constructs used for transient expression studies. Agrotransformants 35S-B3/TLS and 35S-B3/3′UTR were designed, respectively, to express either the 3′ 200-nt region or the entire 3′ UTR. All other features are same those described for panel A. (D) Transient expression and packaging in plants of individual BMV RNAs. N. benthamiana leaves were infiltrated with indicated Agrobacterium cultures, and the total and virion RNAs recovered 4 days postinfiltration were subjected to Northern blot hybridization. Approximately 5 μg of total nucleic acid preparations from agroinfiltrated leaves or 0.5 μg of virion RNA was denatured with formamide-formaldehyde and subjected to 1.2% agarose electrophoresis prior to vacuum blotting to a nylon membrane. The blot was then hybridized with ³²P-labeled riboprobes complementary to the homologous 3′ TLS present on all four BMV RNAs (3′ TLS probe). The position of all four BMV RNAs is shown to the right of each panel. Marker lane B123 represents wt BMV virion RNA recovered from leaves infiltrated with a mixture containing all three wt BMV RNAs. (E) Transient expression and packaging of TLS-less BMV RNAs. The conditions of agroinfiltration and Northern blot analysis of total and virion RNAs are as described above. The blots were hybridized with either a ³²P-labeled 3′ TLS probe or a mixture of riboprobes specific for each of the three genomic RNAs (B1-, B2-, and B4-specific RNAs) (15). Note that since the entire sequence of B4 is present in the 3′ half of B3, riboprobe complementary to the CP ORF sequences (referred to as B4 probe) would detect both B3 and B4. Conditions of hybridization are as described previously (5). The position of four BMV RNAs is shown to the right. wt BMV virion RNA was used as a size marker. For Western blot analysis, total protein extracts from leaves agroinfiltrated independently with the indicated mixture of agrotransformants were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with antibodies prepared against purified BMV (35).

FIG. 2.

(A) Schematic representation of secondary structure of the 3′ 200-nt TLS of BMV RNA3 and 75 nt of cellular tRNA^Tyr. (B) Characteristics of T-DNA construct of sgB4 (35S-B4δ) used for transient expression with authentic 5′ and 3′ termini. In 35S-B4δ, the position of the hepatitis delta ribozyme (δ) is shown. All other features are same those described in the legend of Fig. 1A. Viral and nonviral nucleotide sequences located at the 3′ end are shown by upper- and lowercase, respectively, and the bent arrow represents the ribozyme cleavage site. (C) Packaging competence in vivo of B3ΔTLS and B3^Tyr by transiently expressed CP subunits. N. benthamiana leaves were infiltrated with indicated mixtures of Agrobacterium cultures, and the total and virion RNAs recovered were subjected to Northern hybridization with the indicated riboprobes as described in the legend of Fig. 1D. Marker lane B123 represents wt BMV virion RNA recovered from leaves infiltrated with a mixture containing all three wt BMV RNAs. Western blot analysis of viral CP was performed as described in the legend of Fig. 1D. The positions of B3 and B4 are shown to the right. (D) Packaging efficiency of B3ΔTLS and B3^Tyr. The Northern blots shown in panel C were subjected to quantitation by a Typhoon 9410 PhosphoImager using wt B3 and B4 as internal standards. Histograms represent the relative accumulation of respective total RNA (TR) and virion RNA (VR) for either wt B4 (B4δ), wt B3 (B3), B3^Tyr, or B3ΔTLS.

FIG. 3.

In vivo expression and packaging competence of each of the three replication-defective BMV RNAs. N. benthamiana leaves were infiltrated with indicated mixtures of Agrobacterium cultures (lanes 1 through 5), and the total and virion RNAs recovered were subjected to Northern hybridization with the indicated set of riboprobes. Marker lane B123 represents wt BMV virion RNA recovered from leaves infiltrated with a mixture containing all three wt BMV RNAs.

FIG. 4.

(A) Restoration of the deleted 3′ TLS of B3. N. benthamiana leaves were infiltrated with indicated mixtures of Agrobacterium cultures and the total (T) and virion (V) RNAs recovered were subjected to hybridization with the indicated set of riboprobes as described in the legend of Fig. 1D. Note that detection of sgB4 in total and virion RNAs indicates restoration of a functional 3′ end in B3. In each blot, marker lane B123 represents wt BMV virion RNA recovered from leaves infiltrated with a mixture containing all three wt BMV RNAs.

FIG. 5.

Evidence for restoration of a functional viral TLS in B3 by recombination with transiently expressed 3′ end sequences. N. benthamiana leaves were infiltrated with the indicated mixtures of Agrobacterium cultures (lanes 1 through 4), and the total and virion RNAs recovered were subjected to Northern hybridization with a mixture of riboprobes specific for B1, B2, and B4 as described in the legend of Fig. 1D. Detection of sgB4 in total and virion RNAs indicates restoration of a functional 3′ end in B3. Note that in the first lane, the absence of the TLS in the inoculum failed to restore the 3′ end while packaging of B1ΔΤLS and B2ΔΤLS was not affected. In each blot, marker lane B123 represents wt BMV virion RNA recovered from leaves infiltrated with a mixture containing all three wt BMV RNAs.