RNA-dependent RNA Polymerase Research Articles

Antiviral Potential of Fucoxanthin, an Edible Carotenoid Purified from Sargassum siliquastrum, against Zika Virus.

Considering the lack of antiviral drugs worldwide, we investigated the antiviral potential of fucoxanthin, an edible carotenoid purified from Sargassum siliquastrum, against zika virus (ZIKV) infection. The antiviral activity of fucoxanthin was assessed in ZIKV-infected Vero E6 cells, and the relevant structural characteristics were confirmed using molecular docking and molecular dynamics (MD) simulation. Fucoxanthin decreased the infectious viral particles and nonstructural protein (NS)1 mRNA expression levels at concentrations of 12.5, 25, and 50 µM in ZIKV-infected cells. Fucoxanthin also decreased the increased mRNA levels of interferon-induced proteins with tetratricopeptide repeat 1 and 2 in ZIKV-infected cells. Molecular docking simulations revealed that fucoxanthin binds to three main ZIKV proteins, including the envelope protein, NS3, and RNA-dependent RNA polymerase (RdRp), with binding energies of -151.449, -303.478, and -290.919 kcal/mol, respectively. The complex of fucoxanthin with RdRp was more stable than RdRp protein alone based on MD simulation. Further, fucoxanthin bonded to the three proteins via repeated formation and disappearance of hydrogen bonds. Overall, fucoxanthin exerts antiviral potential against ZIKV by affecting its three main proteins in a concentration-dependent manner. Thus, fucoxanthin isolated from S. siliquastrum is a potential candidate for treating zika virus infections.

Use of Favipiravir in Covid-19 Patients: A Narrative Review

<p><span lang="EN-US">COVID-19 is an acute respiratory disease resulting from the infection of SARS-COV-2 viruses and causes high morbidity, which requires appropriate treatment targets. Favipiravir is an antiviral that selectively inhibits RNA-dependent RNA polymerase (RdRp) of virus. This review aimed to identify several studies that prove the effectiveness and safety of using Favipiravir for COVID-19 patients. The search method used the electronic media PubMed and ScienceDirect with the keywords "Efficacy", "Favipiravir", "Treatment", "Safety", SARS-COV-2", and "Favipiravir induced", accompanied by the addition of the affixes "AND" and "OR" and selection by the publication date starting December 2019. The literature search resulted in eight (8) published articles that met the exclusion and inclusion criteria. The results of the review showed that concurrent administration of Favipiravir and Lopinavir/Ritonavir or Chloroquine, with a dosage of Favipiravir of 3200 mg/day followed by 1200 mg/day each in 2 divided doses, was considered adequate for improving the clinical symptoms of COVID-19 patients with mild-moderate symptoms in early administration. Meanwhile, administering Favipiravir with anti-IL-6 Tocilizumab for patients with severe symptoms showed a fairly good effect. The most frequently reported ADE (adverse drug events) in the use of Favipiravir were hyperuricemia and elevated alanine aminotransferase (ALT) levels. This review concluded that the best clinical response to Favipiravir is shown in COVID-19 patients with mild-to-moderate early symptoms.</span></p>

Genetic detection and analysis of porcine norovirus in pigs farmed in north Vietnam

Norovirus (NoV) causing gastroenteritis symptoms, which has been reported in several hosts, including humans, pigs, and rats. This study was conducted to identify porcine viral infection and to characterize NoV strains from pigs in some provinces in north Vietnam. Totally, 102 fecal samples from diarrheal pigs on farms in six cities and provinces in northern Vietnam during July 2022 to March 2023 were collected. Polymerase chain reaction was used to identify the viral genome. Positive samples were used for nucleotide sequencing of the partial RNA-dependent RNA polymerase gene sequence. Five (4.9 %) positive stool samples were detected from animals farmed in five different farms, with one positive animal identified in each farm. Genetic analysis indicated that nucleotide identity was in the range 97.77–99.62 % among the 5 NoVs in this study. Phylogenetic analysis pointed out that the five NoVs were Genotype II.19 viruses. Genetically, these strains were closely related to porcine NoV strains that were reported in China in 2009.

Exploring the source of TYLCV resistance in Nicotiana benthamiana.

Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most devastating pathogens of tomato, worldwide. It is vectored by the globally prevalent whitefly, Bemisia tabaci, and is asymptomatic in a wide range of plant species that act as a virus reservoir. The most successful crop protection for tomato in the field has been from resistance genes, of which five loci have been introgressed fromwild relatives. Of these, the Ty-1/Ty-3 locus, which encodes an RNA-dependent RNA polymerase 3 (RDR3), has been the most effective. Nevertheless, several TYLCV strains that break this resistance are beginning to emerge, increasing the need for new sources of resistance. Here we use segregation analysis and CRISPR-mediated gene dysfunctionalisation to dissect the differential response of two isolates of Nicotiana benthamiana to TYLCV infection. Our study indicates the presence of a novel non-RDR3, but yet to be identified, TYLCV resistance gene in a wild accession of N. benthamiana. This gene has the potential to be incorporated into tomatoes.

Genome-wide identification of phasiRNAs in Arabidopsis thaliana, and insights into biogenesis, temperature sensitivity, and organ specificity.

The knowledge of biogenesis and target regulation of the phased small interfering RNAs (phasiRNAs) needs continuous update, since the phasiRNA loci are dynamically evolved in plants. Here, hundreds of phasiRNA loci of Arabidopsis thaliana were identified in distinct tissues and under different temperature. In flowers, most of the 24-nt loci are RNA-dependent RNA polymerase 2 (RDR2)-dependent, while the 21-nt loci are RDR6-dependent. Among the RDR-dependent loci, a significant portion is Dicer-like 1-dependent, indicating the involvement of microRNAs in their expression. Besides, two TAS candidates were discovered. Some interesting features of the phasiRNA loci were observed, such as the strong strand bias of phasiRNA generation, and the capacity of one locus for producing phasiRNAs by different increments. Both organ specificity and temperature sensitivity were observed for phasiRNA expression. In leaves, the TAS genes are highly activated under low temperature. Several trans-acting siRNA-target pairs are also temperature-sensitive. In many cases, the phasiRNA expression patterns correlate well with those of the processing signals. Analysis of the rRNA-depleted degradome uncovered several phasiRNA loci to be RNA polymerase II-independent. Our results should advance the understanding on phasiRNA biogenesis and regulation in plants.

Identification and Genome Characterization of a Novel Nege-like Virus Isolated from Aphids (Aphis gossypii) in Yunnan Province.

Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named Aphis gossypii nege-like virus (AGNLV, GenBank: OR880429.1), was isolated from aphids (Aphis gossypii) in Lijiang City, Yunnan, China. AGNLV has a genome sequence of 9258 nt (excluding the polyA tail) encoding three open reading frames (ORFs). ORF1 (7149 nt) encodes a viral methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase. ORF2 (1422 nt) encodes a DiSB-ORF2_chro domain and ORF3 encodes an SP24 domain. The genome sequence of AGNLV shares the highest nucleotide identity of 60.0% and 59.5% with Wuhan house centipede virus 1 (WHCV1) and Astegopteryx formosana nege-like virus (AFNLV), respectively. Phylogenetic analysis based on the RNA-dependent RNA polymerase shows that AGNLV is clustered with other negeviruses and nege-like viruses discovered in aphids, forming a distinct "unclassified clade". Interestingly, AGNLV only encodes three ORFs, whereas AFNLV and WHCV1 have four ORFs. Structure and transmembrane domain predictions show the presence of eight alpha helices and five transmembrane helices in the AGNLV ORF3. Translational enhancement of the AGNLV 5' UTR was similar to that of the 5' UTR of plant viruses. Our findings provide evidence of the diversity and structure of nege-like viruses and are the first record of such a virus from a member of the genus Aphis.

A novel multitargeted self-assembling peptide-siRNA complex for simultaneous inhibition of SARS-CoV-2-host cell interaction and replication

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus's internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-Dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by Cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs veins and arteries endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.

Cross-species transmission of a novel bisegmented orfanplasmovirus in the phytopathogenic fungus Exserohilum rostratum

Mycoviruses have been found in various fungal species across different taxonomic groups, while no viruses have been reported yet in the fungus Exserohilum rostratum. In this study, a novel orfanplasmovirus, namely Exserohilum rostratum orfanplasmovirus 1 (ErOrfV1), was identified in the Exserohilum rostratum strain JZ1 from maize leaf. The complete genome of ErOrfV1 consists of two positive single-stranded RNA segments, encoding an RNA-dependent RNA polymerase and a hypothetical protein with unknown function, respectively. Phylogenetic analysis revealed that ErOrfV1 clusters with other orfanplasmoviruses, forming a distinct phyletic clade. A new family, Orfanplasmoviridae, is proposed to encompass this newly discovered ErOrfV1 and its associated orfanplasmoviruses. ErOrfV1 exhibits effective vertical transmission through conidia, as evidenced by its 100% presence in over 200 single conidium isolates. Moreover, it can be horizontally transmitted to Exserohilum turcicum. Additionally, the infection of ErOrfV1 is cryptic in E. turcicum because there were no significant differences in mycelial growth rate and colony morphology between ErOrfV1-infected and ErOrfV1-free strains. This study represents the inaugural report of a mycovirus in E. rostratum, as well as the first documentation of the biological and transmission characteristics of orfanplasmovirus. These discoveries significantly contribute to our understanding of orfanplasmovirus.

The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses.

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

Using structure prediction of negative sense RNA virus nucleoproteins to assess evolutionary relationships.

Negative sense RNA viruses (NSV) include some of the most detrimental human pathogens, including the influenza, Ebola and measles viruses. NSV genomes consist of one or multiple single-stranded RNA molecules that are encapsidated into one or more ribonucleoprotein (RNP) complexes. These RNPs consist of viral RNA, a viral RNA polymerase, and many copies of the viral nucleoprotein (NP). Current evolutionary relationships within the NSV phylum are based on alignment of conserved RNA-directed RNA polymerase (RdRp) domain amino acid sequences. However, the RdRp domain-based phylogeny does not address whether NP, the other core protein in the NSV genome, evolved along the same trajectory or whether several RdRp-NP pairs evolved through convergent evolution in the segmented and non-segmented NSV genomes architectures. Addressing how NP and the RdRp domain evolved may help us better understand NSV diversity. Since NP sequences are too short to infer robust phylogenetic relationships, we here used experimentally-obtained and AlphaFold 2.0-predicted NP structures to probe whether evolutionary relationships can be estimated using NSV NP sequences. Following flexible structure alignments of modeled structures, we find that the structural homology of the NSV NPs reveals phylogenetic clusters that are consistent with RdRp-based clustering. In addition, we were able to assign viruses for which RdRp sequences are currently missing to phylogenetic clusters based on the available NP sequence. Both our RdRp-based and NP-based relationships deviate from the current NSV classification of the segmented Naedrevirales, which cluster with the other segmented NSVs in our analysis. Overall, our results suggest that the NSV RdRp and NP genes largely evolved along similar trajectories and that even short pieces of genetic, protein-coding information can be used to infer evolutionary relationships, potentially making metagenomic analyses more valuable.

A Potential of Watercress Nasturtium officinale Bioactive Compounds in Inhibiting Infectious Myonecrosis Virus (IMNV) by Targeting RNA-dependent RNA polymerase (RdRp) Virus from Several Countries: In Silico Approach

A Potential of Watercress Nasturtium officinale Bioactive Compounds in Inhibiting Infectious Myonecrosis Virus (IMNV) by Targeting RNA-dependent RNA polymerase (RdRp) Virus from Several Countries: In Silico Approach

RNA-dependent RNA polymerases regulate ascospore discharge through the exonic-sRNA-mediated RNAi pathway.

We found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays important roles in ascospore maturation and ascospore discharge of Fusarium graminearum. These three RNA-dependent RNA polymerases participate in the biogenesis and accumulation of exonic small interference RNA and then regulate ascospore discharge.

Complete genome sequence of a novel totivirus isolated from the leaves of Myrica rubra.

Chinese bayberry is a fruit that is appreciated for its taste. A novel totivirus associated with rolling, disfiguring, chlorotic and vein-clearing symptoms on the leaf apices of Chinese bayberry was identified by transcriptome sequencing and reverse transcription PCR (RT-PCR). The complete genome of the virus was determined to be 4959 nucleotides long, and it contains two open reading frames (ORFs). Its genomic organization is similar to that of previously reported totiviruses. ORF1 encodes a putative coat protein (CP) of 765 aa, and ORF2 encodes an RNA-dependent RNA polymerase (RdRp) of 815 aa. These two putative proteins share 55.1% and 62.6%, amino acid sequence identity, respectively, with the corresponding proteins of Panax notoginseng virus A, respectively. According to the demarcation criteria for totivirus species established by the International Committee on Taxonomy of Viruses (ICTV), the new virus should be considered a member of a new species in the genus totivirus, family Orthototiviridae, which we have tentatively named ''Myrica rubra-associated totivirus'' (MRaTV).

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Fusarium oxysporum f. sp. melonis strain T-SD3.

A novel mitovirus was identified in Fusarium oxysporum f. sp. melonis strain T-SD3 and designated as "Fusarium oxysporum mitovirus 3" (FoMV3). The virus was isolated from diseased muskmelon plants with the typical symptom of fusarium wilt. The complete genome of FoMV3 is 2269 nt in length with a predicted AU content of 61.40% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a polypeptide of 679 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain with a molecular mass of 77.39 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5'-untranslated region (UTR) and 3'-UTR of FoMV3 were predicted to fold into stem-loop structures. BLASTp analysis revealed that the RdRp of FoMV3 shared the highest aa sequence identity (83.85%) with that of Fusarium asiaticum mitovirus 5 (FaMV5, a member of the family Mitoviridae) infecting F. asiaticum, the causal agent of wheat fusarium head blight. Phylogenetic analysis further suggested that FoMV3 is a new member of the genus Unuamitovirus within the family Mitoviridae. This is the first report of a new mitovirus associated with F. oxysporum f. sp. melonis.

HSP90 is part of a protein complex with the L polymerase of Rift Valley fever phlebovirus and prevents its degradation by the proteasome during the viral genome replication/transcription stage.

The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.

Characterization of two novel fusariviruses co-infecting a single isolate of phytopathogenic fungus Botrytis cinerea.

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate. The identified viruses were tentatively namedBotrytis cinerea fusarivirus 9 (BcFV9) and Botrytis cinerea fusarivirus 3a (BcFV3a). Both viruses had a single-segmented (ssRNA) genome having a size of 6424 and 8370 nucleotides encoding two discontinuous open reading frames (ORFs). ORF-1 of both mycoviruses encodes for a polyprotein having a conserved domain of RNA-dependent RNA polymerase (RdRP) and a helicase domain (Hel) which function in RNA replication, while ORF2 encodes a hypothetical protein with an unknown function, respectively. Phylogenetic analysis indicated that BcFV9 made a clade with the genus Alphafusarivirus and BcFV3a fall in the genus Betafusarivirus in the family Fusariviridae. To our knowledge, this is the first report of two fusariviruses identified in isolates of B. cinerea from Pakistan. Both mycoviruses successfully transfected to a compatible strain of B. cinerea (Mst11). A comparison of virus-free (VF) and virus-infected (VI) isogenic lines showed the presence of these viruses was causing hypovirulence in infected strains. Virus-infected strains also had a small lesion size while testing the pathogenicity via apple assay.

Computer-aided designing of a novel multi‑epitope DNA vaccine against severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease caused by the SFTS virus (Dabie bandavirus), which has become a substantial risk to public health. No specific treatment is available now, that calls for an effective vaccine. Given this, we aimed to develop a multi-epitope DNA vaccine through the help of bioinformatics. The final DNA vaccine was inserted into a special plasmid vector pVAX1, consisting of CD8+ T cell epitopes, CD4+ T cell epitopes and B cell epitopes (six epitopes each) screened from four genome-encoded proteins——nuclear protein (NP), glycoprotein (GP), RNA-dependent RNA polymerase (RdRp), as well as nonstructural protein (NSs). To ascertain if the predicted structure would be stable and successful in preventing infection, an immunological simulation was run on it. In conclusion, we designed a multi-epitope DNA vaccine that is expected to be effective against Dabie bandavirus, but in vivo trials are needed to verify this claim.

Lung-targeted delivery of nsp12 siRNAs restores host type I interferon responses

The RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known as nsp12, is crucial to viral replication and spread. Being one of the most conserved proteins of SARS-CoV-2, nsp12 is also a promising therapeutic target for COVID-19. In addition, nsp12 has been reported to suppress the host type I interferon (IFN-I) responses. We used HEK293T cells exogenously expressing nsp12-luciferase to identify siRNAs against nsp12. A series of in vitro and in vivo experiments were employed to examine the effect of RBD-11b-modified cationic liposomes (CLs) on targeted delivery of siRNAs to the mouse lungs. Mice were airway-administered adenovirus to induce the exogenous expression of nsp12 in the mouse lungs, followed by nsp12 siRNAs@RBD-11b-CLs treatment. Nsp12 expression and the activity of the IRF3/IFN-I axis in the lungs were then examined. High-efficiency nsp12 siRNAs were identified and successfully delivered to the mouse lungs by the RBD-11b-modified CLs. The nsp12 siRNAs@RBD-11b-CLs efficiently inhibited the exogenous expression of nsp12 in the mouse lungs. Moreover, nsp12 siRNAs@ RBD-11b-CLs successfully restored the nucleus transport of IRF3, thereby increasing the expression of IFNβ and several IFN effector genes in alveolar cells. We establish a lung targeting system to efficiently deliver siRNAs against SARS-CoV-2 nsp12 and significantly restore host type I interferon responses. This study will provide fresh perspectives and forthcoming approaches for the treatment of COVID-19 and other RNA viruses.

Structural snapshots of phenuivirus cap-snatching and transcription.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.

In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.