Cysteine-Added Mutants of Turnip Yellow Mosaic Virus

Native turnip yellow mosaic virus (TYMV) is relatively unreactive to maleimide agents, indicating few reactive thiol groups on TYMV. In the present study, we aimed to construct TYMV mutants that have reactive cysteine residues on the surface. To this end, we prepared a library of TYMV mutants where the Thr residue at the C-terminus of coat protein (CP) was replaced by a random sequence of six amino acids that included one cysteine. This library was introduced into Nicotiana benthamiana by agroinfiltration. The CP sequence of the TYMV RNA isolated from inoculated leaves was amplified by reverse transcription-PCR and then used to construct a second library. This process was repeated one more time, and the CP sequences of the TYMV RNA in the inoculated leaves were analyzed. Based on the analysis of over 11,000 CP sequences, the Cys mutants representing most abundant TYMV RNAs were constructed. Analysis of the mutants showed that four Cys mutants were nearly comparable to wildtype with respect to CP and viral RNA levels in N. benthamiana. All these mutants were highly reactive to fluoresceine-5-maleimide. This demonstrates that TYMV can be modified to have additional functional groups on the surface that would be useful for drug delivery.

Production and Storage of Virus Simulants

We have examined isolation and identification protocols for three virus simulant candidates to biological warfare agents. MS2 phage, a simulant for yellow fever virus and Hantaan virus, was propagated using as a host an E. coli strain with F pilus. MS2 phage genome was examined by reverse transcription and polymerase chain reaction (RT-PCR). Coat protein of the phage preparation was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. Cydia pomonella granulosis virus (CpGV) is a virus simulant candidate to smallpox virus. CpGV was isolated from a commercialized CpGV pellet. In this study, we developed new isolation and identification protocols for CpGV. One disadvantage of using CpGV is that it is not easy to determine viability of the virus. Here, we have included T4 phage as an alternative. We established a high titer production protocol and developed an easy genome identification protocol that does not require purified phage DNA. Stability of these virus preparations was also examined under various storage conditions. When the virus preparations were not subjected to freeze drying, MS2 phage was most stable when it was stored in liquid nitrogen but unstable at 4℃. In contrast, T4 phage was most stable when it was stored at 4℃. CpGV was stable at −20℃ but not at 4℃. Stability during or after freeze drying was also investigated. The result showed that 70~80% MS2 survived the freeze drying process. In contrast, only about 15% of T4 phage survived during the freeze drying. CpGV was found to be degraded during freeze drying.

Replication of Recombinant Flock House Virus RNA Encapsidated by Turnip Yellow Mosaic Virus Coat Proteins in Nicotiana benthamiana

It was previously observed that recombinant flock house virus (FHV) RNA1 was efficiently packaged into turnip yellow mosaic virus (TYMV), provided that the TYMV coat protein (CP) sequence was present at the 3′-end. FHV RNA encapsidated by TYMV CPs also had a four-nucleotide extension at the 5′-end. Since even a short extension at the 5′- and 3′-ends of FHV RNA1 inhibits replication, we examined whether the recombinant FHV RNA is indeed capable of replication. To this end, we introduced constructs expressing recombinant FHV RNAs into the plant Nicotiana benthamiana. Northern blot analysis of inoculated leaves suggested abundant production of recombinant FHV RNA1 and its subgenomic RNA. This demonstrated that recombinant FHV RNA with terminal extensions at both ends was competent for replication. We also showed that the recombinant FHV RNA can express the reporter gene encoding enhanced green fluorescent protein.

N-terminal Extension of Coat Protein of Turnip Yellow Mosaic Virus has Variable Effects on Replication, RNA Packaging, and Virion Assembly Depending on the Inserted Sequence

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus composed of 20 kDa single coat proteins. In this study, we modified the TYMV coat protein (CP) ORF by inserting an oligonucleotide linker corresponding to T7, HSV, Tat, (Arg)9, or (RxR)4 peptide at the 5'-end of the CP ORF and examined its effect on replication, RNA packaging, and virion assembly. The results showed that the constructs containing (Arg)9 and (RxR)4 sequences were barely capable of replication. The TYMV constructs containing T7 and Tat peptide produced virions that co-migrated with wild-type virions. However, the insertion of T7 and Tat sequences impaired genomic RNA (gRNA) accumulation and packaging, respectively. When only the CP gene was expressed, CPs with (Arg)9 or (RxR)4 successfully produced virus-like particles whose mobility was comparable to that of wild type. In the case of CP having a HSV tag, the virion band was not detected, although a sufficient amount of CP was produced. This indicates that CP with the HSV tag failed to assemble into virions. Overall, the results suggest that TYMV replication, RNA packaging and virion assembly are strongly influenced by the insertion sequence.

Genome Size Constraint in Replication and Packaging of Turnip Yellow Mosaic Virus

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.

Read-through Mutation in the Coat Protein ORF Suppresses Turnip Yellow Mosaic Virus Subgenomic RNA Accumulation

We have previously observed that a sequence in coat protein (CP) ORF of Turnip yellow mosaic virus (TYMV) is required for efficient replication of the virus. The sequence was predicted to take a stem-loop structure, thus termed SL2. While examining various SL2 mutants, we observed that all the modifications resulting in extension of translation beyond the CP ORF significantly suppressed subgenomic RNA accumulation. The genomic RNA level, in contrast, was not affected. Introduction of an in-frame stop codon in the CP ORF of these constructs restored the level of subgenomic RNA. Overall, the results suggest that the read-through makes the subgenomic RNA unstable.

Characterization of a Replication Element in the Coat Protein ORF of Turnip Yellow Mosaic Virus

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus that has a single 6.3 kb positive-strand RNA as a genome. Previously, it was observed that the recombinant construct TY-eGFP2, where an eGFP gene was inserted at the position downstream of the coat protein (CP) ORF of TYMV genome, barely replicated. The inhibition of replication was relieved by insertion of an additional copy of the 3' quarter of the CP ORF after the foreign sequence. In this study, we have examined if the 3' quarter of the CP ORF contains any replication elements. M-fold analysis predicted three stem-loop structures in this region. Analysis of the TY-eGFP2 constructs containing one or two of these stem-loop structures indicates that the secondary structure predicted in the region between nt-6139 and nt-6181, termed SL2, is essential for TYMV replication. The critical role of SL2 was confirmed by the observation that deletion of the 3' quarter of the CP ORF from the wild-type TYMV genome nearly abolished replication and that insertion of SL2 into the deletion mutant restored the replication. Mutations disrupting the stem of SL2 greatly reduced viral RNA replication, indicating that the secondary structure is essential for the enhancing activity.

A Sequence in Coat Protein Open Reading Frame Is Required for Turnip Yellow Mosaic Virus Replication

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA genome. Information for TYMV replication is limited, except that the 3'-terminal sequence and 5'-untranslated region are required for genome replication. When a foreign sequence was inserted at the position upstream of the coat protein (CP) open reading frame (ORF), replication of the recombinant TYMV was comparable to wild type, as long as an RNAi suppressor was provided. In contrast, when the foreign sequence was inserted between the CP ORF and the 3'-terminal tRNA-like structure, replication of the recombinant virus was not detected. This result suggests that the CP ORF contains an essential replication element which should be appropriately spaced with respect to the 3'-end. Analysis of TYMV constructs containing a part or a full additional CP ORF indicates that the 3' quarter of the CP ORF is required for TYMV replication.