RNA Encapsidation Research Articles

Role of tRNA-like structures in controlling plant virus replication

Transfer RNA-like structures (TLSs) that are sophisticated functional mimics of tRNAs are found at the 3′-termini of the genomes of a number of plant positive strand RNA viruses. Three natural aminoacylation identities are represented: valine, histidine, and tyrosine. Paralleling this variety in structure, the roles of TLSs vary widely between different viruses. For Turnip yellow mosaic virus, the TLS must be capable of valylation in order to support infectivity, major roles being the provision of translational enhancement and down-regulation of minus strand initiation. In contrast, valylation of the Peanut clump virus TLS is not essential. An intermediate situation seems to exist for Brome mosaic virus, whose RNAs 1 and 2, but not RNA 3, need to be capable of tyrosylation to support infectivity. Other known roles for certain TLSs include: (i) the recruitment of host CCA nucleotidyltransferase as a telomerase to maintain intact 3′ CCA termini, (ii) involvement in the encapsidation of viral RNAs, and (iii) presentation of minus strand promoter elements for replicase recognition. In the latter role, the promoter elements reside within the TLS but are not functionally dependent on tRNA mimicry. The phylogenetic distribution of TLSs indicates that their evolutionary history includes frequent horizontal exchange, as has been observed for protein-coding regions of plant positive strand RNA viruses.

Distinct viral determinants for the packaging of human cytidine deaminases APOBEC3G and APOBEC3C

Human APOBEC3G and other APOBEC3 cytidine deaminases inhibit a variety of retroviruses, including Vif-deficient HIV-1. These host proteins are packaged into viral particles and inhibit the replication of virus in new target cells. A3G and A3F are known to be efficiently packaged into HIV-1 virions by binding to 7SL RNA through the Gag NC domain; however, the packaging mechanisms of other APOBEC3 proteins are poorly defined. We have now demonstrated that APOBEC3C (A3C) can be efficiently packaged into HIV-1 virions that are deficient for viral genomic RNA. Inhibition of the encapsidation of 7SL RNA into HIV-1 virions blocked the packaging of A3G, but not A3C. While the NC domain is required for efficient packaging of A3G, deletion of this domain had little effect on A3C packaging into HIV-1 Gag particles. A3C interacted with HIV-1 Gag which was MA domain-dependent and RNA-dependent. Deletion of the MA domain of HIV-1 Gag inhibited A3C but not A3G packaging into HIV-1 Gag particles. Thus, A3G and A3C have evolved to use distinct mechanisms for targeting retroviruses.

Comparison of Serum Humoral Responses Induced by Oral Immunization with the Hepatitis B Virus Core Antigen and the Cholera Toxin B Subunit

The hepatitis B virus core (HBc) virus-like particle (VLP) is known as one of the most immunogenic antigens and carrier vehicles in different immunization strategies. Recent findings are suggesting the potential of the HBc VLPs as an oral immunogen. Here, we focus on the induction of serum humoral responses by oral administration of HBc VLPs in preparations substantially free of lipopolysaccharide and immunomodulating encapsidated RNA. The full-length HBc antigen was used, because the C-terminal arginine-rich tail may contribute to the immunogenicity of the antigen as the region is involved in cell surface heparan sulfate binding and internalization of the protein. Serum antibody levels and isotypes were determined following oral administration of the HBc VLPs with the perspective of using the HBc VLP as an immunostimulatory and carrier molecule for epitopes of blood-borne diseases in oral immunization vaccination strategies. Following oral administration of the HBc VLP preparations to mice, a strong serum humoral response was induced with mainly immunoglobulin G2a (IgG2a) antibodies, pointing toward a Th1 response which is essential in the control of intracellular pathogens. Intraperitoneal immunization with the HBc VLP induced a stronger, mixed Th1/Th2 response. Finally, a comparison was made with the induced serum humoral response following oral administration of the recombinant cholera toxin B pentamer, a commonly used oral immunogen. These immunizations, in contrast, induced predominantly antibodies of the IgG1 isotype, indicative of a Th2 response. These data suggest that the HBc VLP can be an interesting carrier molecule in oral vaccine development.

Functional complementation of nucleocapsid and late domain PTAP mutants of human immunodeficiency virus type 1 during replication

During human immunodeficiency virus type 1 (HIV-1) assembly, the nucleocapsid (NC) and the PTAP motif in p6 of Gag play important roles in RNA encapsidation and virus release, respectively. We have previously demonstrated that functional complementation occurs between an NC mutant and a PTAP mutant to rescue viral replication. In this report, we examined the amounts of functional NC and PTAP motif that are required during virus replication. When NC and PTAP mutants were coexpressed at 5:1, 5:5, and 1:5 ratios, virus titers were rescued at 5%, 51%, and 86% of the wild-type level, respectively. These results indicate that HIV-1 requires a small amount of functional PTAP motif but far more functional NC to complete efficient replication. Further analyses reveal that RNA packaging can be significantly rescued in viruses containing a small amount of functional NC. However, most of the NC proteins must be functional to generate the wild-type level of R-U5 DNA product. Once the R-U5 product is generated, viruses containing half of the functional NC can complete reverse transcription and DNA integration at near-wild-type efficiency. These results define the quantitative requirements of NC and p6 during HIV-1 replication and provide insights into the requirement for the development of anti-HIV strategies using NC and p6 as targets.

Role of Sindbis Virus Capsid Protein Region II in Nucleocapsid Core Assembly and Encapsidation of Genomic RNA

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.

Reverse transcriptase activity of hepatitis B virus polymerase in eukaryotic cell extracts in vitro

In hepadnaviruses, reverse transcription is primed by the viral reverse transcriptase (RT) and requires the specific interaction between the RT and the viral RNA encapsidation signal termed e. To study the activity of the RT in vitro, the current procedure uses in vitro translated duck hepatitis B virus polymerase, but not the hepatitis B virus polymerase itself, in the rabbit reticulocyte lysate expression system. Here, the hepatitis B virus (HBV) polymerase has been successfully expressed in a translational extract that was obtained from monolayer human hepatocyte cells HuH-7. The translated polypeptide retained the RNA-directed polymerase (reverse transcriptase) activity on the viral RNA template containing the e signal. We suggest that the reverse transcription event of the viral RNA coding for the polymerase and containing an e structure is concomitant to the translation of the viral polymerase to the messenger RNA. In contrast to the duck polymerase, only a fraction of the reverse transcribed complementary DNA (cDNA) was covalently bound to the HBV polymerase in this system. When the e signal was missing on the mRNA, the translated full-length HBV polymerase could not reverse transcribe the viral RNA template. A truncated HBV polymerase that was lacking the YMDD catalytic active site for the initiation of reverse transcription was unable to reverse transcribe the viral mRNA template containing the e signal. The reverse transcription activity could also be partially inhibited by employing nucleoside analogues, such as 2'-3'-dideoxy-3'-thiacytidine (3TC; lamivudine) in the expression system. The procedure described here provides a method for the in vitro screening of new anti-HBV compounds directed against wild- type and mutants of this crucial viral protein, the HBV polymerase, without the use of animals (ducks) or animal extracts (rabbit reticulocyte lysate).

Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity

BackgroundRetroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5' UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation in vivo and its implication in viral replication.ResultsUsing a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores.ConclusionIn addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target.

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

Role of Intermolecular Interactions of Vesicular Stomatitis Virus Nucleoprotein in RNA Encapsidation

The crystal structure of the vesicular stomatitis virus nucleoprotein (N) in complex with RNA reveals extensive and specific intermolecular interactions among the N molecules in the 10-member oligomer. What roles these interactions play in encapsidating RNA was studied by mutagenesis of the N protein. Three N mutants intended for disruption of the intermolecular interactions were designed and coexpressed with the phosphoprotein (P) in an Escherichia coli system previously described (T. J. Green et al., J. Virol. 74:9515-9524, 2000). Mutants N (Delta1-22), N (Delta347-352), and N (320-324, (Ala)(5)) lost RNA encapsidation and oligomerization but still bound with P. Another mutant, N (Ser290-->Trp), was able to form a stable ring-like N oligomer and bind with the P protein but was no longer able to encapsidate RNA. The crystal structure of N (Ser290-->Trp) at 2.8 A resolution showed that this mutant can maintain all the same intermolecular interactions as the wild-type N except for a slight unwinding of the N-terminal lobe. These results suggest that the intermolecular contacts among the N molecules are required for encapsidation of the viral RNA.

Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein

The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

Interaction of Vesicular Stomatitis Virus P and N Proteins: Identification of Two Overlapping Domains at the N Terminus of P That Are Involved in N 0 -P Complex Formation and Encapsidation of Viral Genome RNA

The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N(0)). The N(0)-P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione S-transferase fused to the N-terminal 40 aa by itself is able to form the N(0)-P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P (PDelta1-10) lacking the N-terminal 10 aa which is capable of forming the N(0)-P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcription-reconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (PDeltaII+III). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N(0)-P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation.

2b ORFs encoded by subgroup IB strains of cucumber mosaic virus induce differential virulence on Nicotiana species

Cucumber mosaic virus (CMV)-encoded 2b protein from subgroup IA or subgroup II was shown to be a determinant of virulence in many solanaceous hosts. In this study, the virulence of 2b proteins from subgroup IB strains was analysed using four intraspecies hybrid viruses, which were generated by precise replacement of the 2b open reading frame (ORF) in subgroup IA strain Fny-CMV with the 2b ORFs of four subgroup IB strains, Cb7-CMV, PGs-CMV, Rad35-CMV and Na-CMV, generating FCb7(2b)-CMV, FPGs(2b)-CMV, FRad35(2b)-CMV and FNa(2b)-CMV, respectively. FCb7(2b)-CMV was more virulent than Fny-CMV, and was similar in phenotype to its parental virus Cb7-CMV on the three Nicotiana species tested. FNa(2b)-CMV also was virulent on these host species, equivalent to Fny-CMV or Na-CMV. However, FRad35(2b)-CMV only caused mild mosaic or undetectable symptoms on all the host species tested, and was less virulent than Fny-CMV or Rad35-CMV. FPGs(2b)-CMV infected all the host species systemically, and induced either mosaic or barely visible symptoms, demonstrating that the inability of PGs-CMV to infect these three Nicotiana species was not due to its 2b protein. The diverse virulence was shown to be mediated by the 2b proteins rather than the C-terminal overlapping parts of the 2a proteins, and was associated with the level of viral progeny RNA accumulation in systemically infected leaves, but not with the rate of long-distance viral movement in host plants. Through analysis of encapsidation of viral RNAs, there was an apparent correlation between the virulence and the high level of encapsidated RNA 2 in virions of Fny-CMV, FCb7(2b)-CMV and FNa(2b)-CMV.

A novel virus isolated from the aphid Brevicoryne brassicae with similarity to Hymenoptera picorna-like viruses

A novel virus, Brevicoryne brassicae virus (BrBV), has been identified in the cabbage aphid using a method based on the random amplification of encapsidated RNA. The complete sequence of the RNA genome of BrBV has been determined. The positive-strand genomic RNA is 10 161 nt, excluding the 3' poly(A) tail, and contains a single open reading frame (positions 793-9744) encoding a putative polyprotein of 2983 aa. The N-terminal part of the polyprotein shows similarity with the structural proteins of iflaviruses. The C-terminal part possesses consensus sequences of the helicase, cysteine protease and RNA-dependent RNA polymerase similar to those of iflaviruses and other picorna-like viruses. The highest sequence similarity observed was with iflaviruses from honeybee and an endoparasitic wasp. Replication and transmission of BrBV was not dependent on endoparasitic wasp infestation of the aphids.

Development of a novel recombinant encapsidated RNA particle: Evaluation as an internal control for diagnostic RT-PCR

This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot-and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants ( Vigna unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious.

The phenylpropenamide derivative AT-130 blocks HBV replication at the level of viral RNA packaging

Nucleos(t)ide analogue antiviral therapy for chronic hepatitis B has proven to be effective in the short term but the frequent development of resistance limits its clinical utility. Agents targeting other stages of viral replication are needed in order to develop improved combination therapies. The phenylpropenamide derivatives AT-61 and AT-130 have been shown to inhibit HBV replication in vitro, but the mechanism of action of these compounds remains undefined. The aim of this study was to determine the mechanism of action of AT-130, a non-nucleoside inhibitor of HBV in several in vitro models of replication. These studies found that AT-130 inhibited HBV DNA replication in hepatoma cells but had no effect on viral DNA polymerase activity or core protein translation. Total HBV RNA production was also unaffected in the presence of the drug whilst the amount of encapsidated RNA was significantly reduced, thereby inhibiting subsequent viral reverse transcription. These studies have established that the inhibition of HBV genome replication by a non-nucleoside analogue acting at the level of viral encapsidation and packaging is a potentially useful strategy for future therapeutic drug development in the management of chronic hepatitis B.

Potato virus X: structure, disassembly and reconstitution

SUMMARY This paper summarizes some structural characteristics of Potato virus X (PVX), the flexuous filamentous plant potexvirus. A model of PVX coat protein (CP) tertiary structure in the virion proposed on the basis of tritium planigraphy combined with predictions of the protein tertiary structure is described. A possible role of glycosylation and phosphorylation in the CP structure and function is discussed. Two forms of PVX virion disassembly are discussed: (i) the virion co-translational disassembly after PVX CP in situ phosphorylation and (ii) disassembly of PVX triggered by different factors after linear destabilization of the virion by binding of the PVX-coded movement protein (TGBp1) to one end of the polar CP-helix. Special emphasis was placed on a translational activation of encapsidated PVX RNA and rapid disassembly of TGBp1-PVX complexes into free RNA and CP. The results of experiments on the PVX CP repolymerization and PVX reconstitution are considered. In particular, the products assembled from PVX RNA, CP and TGBp1 were examined. Single-tailed particles were found with a helical, head-like structure consisting of helically arranged CP subunits located at the 5'-tail of RNA; the TGBp1 was bound to the end of the head. Translatable 'RNA-CP-TGBp1' complexes may represent the transport form of the PVX infection.

Analysis of the contribution of cellular and viral RNA to the packaging of APOBEC3G into HIV-1 virions

BackgroundEfficient incorporation of the cellular cytidine deaminase APOBEC3G (APO3G) into HIV-1 virions is necessary for its antiviral activity. Even though cellular RNAs are known to be non-specifically incorporated into virus particles, we have previously found that encapsidation of APO3G into HIV-1 virions is specifically enhanced by viral genomic RNA. Intracellularly, APO3G was found to form large RNA-protein complexes involving a variety of cellular RNAs. The goal of this study was to investigate the possible contribution of host RNAs recently identified in intracellular APO3G ribonucleoprotein complexes to APO3G's encapsidation into HIV-1 virions.ResultsOur results show that 7SL RNA, a component of signal recognition particles, and hY1, hY3, hY4, hY5 RNAs were present in intracellular APO3G complexes and were packaged into HIV-1 particles lacking viral genomic RNA unlike APO3G, which was not packaged in significant amounts into genomic RNA-deficient particles. These results indicate that packaging of 7SL or hY RNAs is not sufficient for the packaging of APO3G into HIV-1 virions. We also tested the encapsidation of several other cellular RNAs including β-actin, GAPDH, α-tubulin, and small nuclear RNAs and determined their effect on the packaging of APO3G into nascent virions. Again, we were unable to observe any correlation between APO3G encapsidation and the packaging of any of these cellular RNAs.ConclusionThe results from this study support our previous conclusion that viral genomic RNA is a critical determinant for APO3G incorporation into HIV-1 virions. While most cellular RNAs tested in this study were packaged into viruses or virus-like particles we failed to identify a correlation between APO3G encapsidation and the packaging of these cellular RNAs.

Critical role of the 36-nucleotide insertion in hepatitis B virus genotype G in core protein expression, genome replication, and virion secretion.

Frequent coinfection of hepatitis B virus genotype G with genotype A suggests that genotype G may require genotype A for replication or transmission. In this regard, genotype G is unique in having a 12-amino-acid extension in the core protein due to a 36-nucleotide insertion near the core gene translation initiation codon. The insertion alters base pairing in the lower stem of the pregenome encapsidation signal, which harbors the core gene initiator, and thus has the potential to affect both core protein translation and pregenomic RNA encapsidation. Genotype G is also unusual for possessing two nonsense mutations in the precore region, which together with the core gene encode a secreted nonstructural protein called hepatitis B e antigen (HBeAg). We found that genotype G clones were indeed incapable of HBeAg expression but were competent in RNA transcription, genome replication, and virion secretion. Interestingly, the 36-nucleotide insertion markedly increased the level of core protein, which was achieved at the level of protein translation but did not involve alteration in the mRNA level. Consequently, the variant core protein was readily detectable in patient blood. The 12-amino-acid insertion also enhanced the genome maturity of secreted virus particles, possibly through less efficient envelopment of core particles. Cotransfection of genotypes G and A did not lead to mutual interference of genome replication or virion secretion. Considering that HBeAg is an immunotolerogen required for the establishment of persistent infection, its lack of expression rather than a replication defect could be the primary determinant for the rare occurrence of genotype G monoinfection.

Molecular biology of potexviruses: recent advances

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5' non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3' NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr-R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

A putative new domain target for anti‐hepatitis B virus: Residues flanking hepatitis B virus reverse transcriptase residue 306 (rtP306)

Previous work showed that conservation of proline at residue 306 (rtP306) of hepatitis B virus (HBV) reverse transcriptase (RT) is crucial for virus replication and encapsidation of pregenomic RNA (pgRNA). In this study, the functions of residues flanking rtP306 in HBV RT (rtG304, rtY305, rtA307, rtL308 and rtL311) are presented. Alanine or phenylalanine was used to substitute these residues by constructing site-directed mutants which were used to transfect Huh-7 cells. Replication competencies and encapsidation efficiencies were compared between the mutants and the parental viral strain. Substitutions at these residues resulted in marked decrease of replication competency, which was confirmed by Southern blot hybridization of HBV DNA isolated from intracytoplasmic core particles, and trans-complementation between a non-replicative defective mutant and corresponding RT mutants. Impaired pgRNA encapsidation efficiency of these mutants was shown as the major mechanism for decreased replication efficiency. Since residues from rt304 to rt311 are highly conserved among genotypes A-H HBV strains, results suggest that rt304 to rt311 in HBV RT may serve as a putative anti-HBV new target domain.