Identification of Arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein

Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement. The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins. Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants, and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7.     

Second-site suppressor mutations assist in studying the function of the 3'' noncoding region of turnip yellow mosaic virus RNA.

The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57----U mutation in the tRNA-like structure was a strong suppressor of the C-55----A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107----C mutation was a strong second-site suppressor of the U-96----G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G + 92----A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A + 92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A + 92/G-96 double mutant in plants.     

Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.     

The plant gene CCD1 selectively blocks cell death during the hypersensitive response to Cauliflower mosaic virus infection

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.     

Genetic interactions between an essential 3' cis-acting RNA pseudoknot, replicase gene products, and the extreme 3' end of the mouse coronavirus genome

The upstream end of the 3' untranslated region (UTR) of the mouse hepatitis virus genome contains two essential and overlapping RNA secondary structures, a bulged stem-loop and a pseudoknot, which have been proposed to be elements of a molecular switch that is critical for viral RNA synthesis. It has previously been shown that a particular six-base insertion in loop 1 of the pseudoknot is extremely deleterious to the virus. We have now isolated multiple independent second-site revertants of the loop 1 insertion mutant, and we used reverse-genetics methods to confirm the identities of suppressor mutations that could compensate for the original insertion. The suppressors were localized to two separate regions of the genome. Members of one class of suppressor were mapped to the portions of gene 1 that encode nsp8 and nsp9, thereby providing the first evidence for specific interactions between coronavirus replicase gene products and a cis-acting genomic RNA element. The second class of suppressor was mapped to the extreme 3' end of the genome, a result which pointed to the existence of a direct base-pairing interaction between loop 1 of the pseudoknot and the genomic terminus. The latter finding was strongly supported by phylogenetic evidence and by the construction of a deletion mutant that reduced the 3' UTR to its minimal essential elements. Taken together, the interactions revealed by the two classes of suppressors suggest a model for the initiation of coronavirus negative-strand RNA synthesis.     

Genetic Analysis of Staphylococcal Nuclease: Identification of Three Intragenic "Global" Suppressors of Nuclease-minus Mutations

A collection of 77 unique missense mutations distributed across the gene encoding staphylococcal nuclease (nuc) has been assembled. These mutations were induced by random gap misrepair mutagenesis of the cloned gene and were identified in E. coli transformants expressing reduced levels of nuclease activity. Four nuc- mutations which alter amino acid residues at positions outside of the active site region of the enzyme were submitted to a second round of mutagenesis, and characterization of several independent NUC+ isolates lead to the identification of three second-site suppressor mutations within the protein-coding sequence of the nuc gene. On separation from the mutation originally suppressed and recombination with a number of other nuc- mutations, all three suppressors displayed the property of "global" suppression, i.e., phenotypic suppression of the nuclease-minus character of multiple different alleles. A simple and generally applicable strategy was used to obtain efficient homologous recombination between plasmids for purposes of mapping nuc- mutations, mapping second-site suppressors and constructing double mutant combinations from pairs of single mutations.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor

The M(3) muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M(3) muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M(3) receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.     

A herpes simplex virus type 1 gamma34.5 second-site suppressor mutant that exhibits enhanced growth in cultured glioblastoma cells is severely attenuated in animals

We describe here the neurovirulence properties of a herpes simplex virus type 1 gamma34.5 second-site suppressor mutant. gamma34.5 mutants are nonneurovirulent in animals and fail to grow in a variety of cultured cells due to a block at the level of protein synthesis. Extragenic suppressors with restored capacity to replicate in cells that normally do not support the growth of the parental gamma34.5 deletion mutant have been isolated. Although the suppressor virus reacquires the ability to grow in nonpermissive cultured cells, it remains severely attenuated in mice and is indistinguishable from the mutant gamma34.5 parent virus at the doses investigated. Repairing the gamma34.5 mutation in the suppressor mutant restores neurovirulence to wild-type levels. These studies illustrate that (i) the protein synthesis and neurovirulence defects observed in gamma34.5 mutant viruses can be genetically separated by an extragenic mutation at another site in the viral chromosome; (ii) the extragenic suppressor mutation does not affect neurovirulence; and (iii) the attenuated gamma34.5 mutant, which replicates poorly in many cell types, can be modified by genetic selection to generate a nonpathogenic variant that regains the ability to grow robustly in a nonpermissive glioblastoma cell line. As this gamma34.5 second-site suppressor variant is attenuated and replicates vigorously in neoplastic cells, it may have potential as a replication-competent, viral antitumor agent.     

Effects of point mutations in the readthrough domain of the beet western yellows virus minor capsid protein on virus accumulation in planta and on transmission by aphids

Point mutations were introduced into or near five conserved sequence motifs of the readthrough domain of the beet western yellows virus minor capsid protein P74. The mutant virus was tested for its ability to accumulate efficiently in agroinfected plants and to be transmitted by its aphid vector, Myzus persicae. The stability of the mutants in the agroinfected and aphid-infected plants was followed by sequence analysis of the progeny virus. Only the mutation Y201D was found to strongly inhibit virus accumulation in planta following agroinfection, but high accumulation levels were restored by reversion or pseudoreversion at this site. Four of the five mutants were poorly aphid transmissible, but in three cases successful transmission was restored by pseudoreversion or second-site mutations. The same second-site mutations in the nonconserved motif PVT(32-34) were shown to compensate for two distinct primary mutations (R24A and E59A/D60A), one on each side of the PVT sequence. In the latter case, a second-site mutation in the PVT motif restored the ability of the virus to move from the hemocoel through the accessory salivary gland following microinjection of mutant virus into the aphid hemocoel but did not permit virus movement across the epithelium separating the intestine from the hemocoel. Successful movement of the mutant virus across both barriers was accompanied by conversion of A59 to E or T, indicating that distinct features of the readthrough domain in this region operate at different stages of the transmission process.     

Analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted RNA recombination.

The Alb4 mutant of the coronavirus mouse hepatitis virus (MHV) is both temperature sensitive and thermolabile owing to a deletion in the gene encoding its nucleocapsid (N) protein. The deletion removes 29 amino acids that constitute a putative spacer region preceding the carboxyl-terminal domain of the protein. As a step toward understanding the structure and function of the MHV N protein, we isolated multiple independent revertants of Alb4 that totally or partially regained the ability to form large (wild-type-sized) plaques at the nonpermissive temperature. The N proteins of these revertant viruses concomitantly regained the ability to bind to RNA in vitro at a temperature that was restrictive for RNA binding by Alb4 N protein. Sequence analysis of the N genes of the revertants revealed that each contained a single second-site point mutation that compensated for the effects of the deletion. All reverting mutations were clustered within a stretch of 40 amino acids centered some 80 residues on the amino side of the Alb4 deletion, within a domain to which the RNA-binding activity of N had been previously mapped. By means of a targeted RNA recombination method that we have recently developed, two of the reverting mutations were introduced into a wild-type MHV genomic background. The resulting recombinants were stable and showed no gross phenotypic differences from the wild type. A detailed analysis of one, however, revealed that it was at a selective disadvantage with respect to the wild type.     

Long-distance movement,virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families

RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the gammab gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the gammab protein may be a long-distance movement factor and have antisilencing activity. This was shown for gammab proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, gammab and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the gammab cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus gammab proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.     

Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication.

Base substitution of the ori region of simian virus 40 leads to plaque morphology mutants with markedly decreased DNA replication. Second-site mutations within the simian virus 40 T antigen gene suppress the plaque phenotype and replication defect of base-substituted ori mutants. Two second-site mutations have been mapped to a small segment of the T antigen gene, just beyond the distal splice junction. DNA sequence analysis revealed a single missense change in this segment of the T antigen gene of each of these second-site revertants, leading to a change in codon 157 in one case and codon 166 in the other. The mutant T antigens displayed relaxed specificity for the ori signal, i.e., they can function with several variously modified ori sequences, including those with small nucleotide deletions or insertions that are inactive for replication when coupled with wild-type T antigen. Thus a region of T antigen has been identified that appears to be intimately involved in vivo in binding to the ori sequence to initiate viral DNA replication.     

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation,virus accumulation,and aphid transmission

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.     

Suppressors of a host range mutation in the rabbitpox virus serpin SPI-1 map to proteins essential for viral DNA replication

The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.     

Restoration of mRNA splicing by a second-site intragenic suppressor in the T4 ribonucleotide reductase (small subunit) self-splicing intron

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base self-splicing intron which is closely related to other group I introns of T4 and eukaryotes. Thirty-one mutants causing splicing defects in the nrdB intron were isolated. Twenty-three EMS-induced revertants for these 31 primary mutants were isolated by the strategic usage of the white halo plaque phenotype. We mapped these revertants by marker rescue using subclones of the nrdB gene. Some of these second-site mutations mapped to regions currently predicted by the secondary structure model of the nrdB intron. One of these suppressor mutants (nrdB753R) was found to be intragenic by marker rescue with the whole nrdB gene. However, this mutation failed to map within the nrdB intron. Splicing assays showed that this pseudorevertant restored splicing proficiency of the nrdB primary mutation to almost wild-type conditions. This is the first example of a mutation within the exons of a gene containing a self-splicing intron that is capable of restoring a self-splicing defect caused by a primary mutation within the intron. In addition, two other suppressor mutations are of interest (nrdB429R and nrdB399R). These suppressors were able to restore their primary 5' defect but in turn create a 3' splicing defect. Both of these revertants mapped in different regions of the intron with respect to their primary mutations.