Sindbis virus nsP1 functions in negative-strand RNA synthesis.

A mutation at nucleotide 1101 of Sindbis virus ts11 nsP1 caused temperature-sensitive negative-strand synthesis and suppressed the 24R phenotype, which is caused by a mutation in nsP4. Nonstructural proteins synthesized and accumulated by ts11 at 40 degrees C did not cause the reactivation of negative-strand synthesis upon return to 30 degrees C and did not prevent the formation of new replication complexes at 30 degrees C.     

Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication

Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication. Two cis elements controlling RNA3 production were found. A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides [nt] 2282 to 2777). A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239. Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element. Two distinct RNA1 replication elements (RE) were mapped within the 3' quarter of RNA1: the intRE (nt 2322 to 2501) and the 3'RE (nt 2735 to 3011). The 3'RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression. In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production.     

Role of the alfalfa mosaic virus methyltransferase-like domain in negative-strand RNA synthesis

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.     

Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.     

Nonhomologous recombination in human cells.

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.     

Translating ribosomes inhibit poliovirus negative-strand RNA synthesis

Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation "freeze" ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.     

Cotranslational disassembly of flock house virus in a cell-free system.

Intact, purified particles of the nodaviruses flock house virus and nodamura virus that were either transfected into cells that were resistant to infection or introduced into in vitro translation systems directed the synthesis of viral proteins. We infer that direct interaction of these nodavirus particles with cytoplasmic components mediated virion disassembly that resulted in release of the viral RNA.     

A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells

The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.     

The 5' untranslated region of alfalfa mosaic virus RNA 1 is involved in negative-strand RNA synthesis

The three genomic RNAs of alfalfa mosaic virus each contain a unique 5' untranslated region (5' UTR). Replacement of the 5' UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5' stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5' stem-loop structure is present in RNA 2 but not in RNA 3.     

Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two messenger-sense RNA molecules, both of which are encapsidated in the same particle. RNA1 (3.1 kb) encodes proteins required for viral RNA replication; RNA2 (1.4 kb) encodes protein alpha (43 kDa), the precursor of the coat protein. When Spodoptera frugiperda cells were infected with a recombinant baculovirus containing a cDNA copy of RNA2, coat protein alpha assembled into viruslike precursor particles (provirions) that matured normally by autocatalytic cleavage of protein alpha into polypeptide chains beta (38 kDa) and gamma (5 kDa). The particles were morphologically indistinguishable from authentic FHV and contained RNA derived from the coat protein message. These results showed that RNA1 was required neither for virion assembly nor for maturation of provirions. Expression of mutants in which Asn-363 at the beta-gamma cleavage site of protein alpha was replaced by either aspartate, threonine, or alanine resulted in assembly of particles that were cleavage defective. For two of the mutants, unusual structural features were observed after preparation for electron microscopy. Particles containing Asp at position 363 were labile and showed a strong tendency to break into half-shells. Particles in which Asn-363 was replaced by Ala displayed a distinct hole in an otherwise complete shell. The third mutant, containing Thr at position 363, was indistinguishable in morphology from authentic FHV.     

Membrane partitioning of the cleavage peptide in flock house virus

Membrane translocation of the ssRNA genome of nodaviruses has been proposed to be mediated by direct lipid-protein interactions between a postassembly autocatalytic cleavage product from the capsomere and the target membrane. We have recently shown that the 21-residue Met-->Nle variant of the N-terminal helical domain (denoted gamma(1)) of the cleavage peptide in flock house nodavirus increases membrane permeability to hydrophilic solutes and can alter both membrane structure and function, suggesting the possibility of peptide-triggered disruption of the endosomal membrane as a prelude to viral uncoating in the host cytoplasm. Elucidation of partitioning energetics would allow an assessment of the likelihood of this mechanism. We report herein complete thermodynamic characterization of the partitioning of gamma(1) to phospholipids by lipid-peptide titrations following changes in ellipticity, fluorescence signature, or calorimetric response. These experiments revealed a partitioning energy comparable to natural membrane-active peptide toxins, suggesting that the proposed mechanism may be possible. Additionally, a novel switch in the balance of partitioning forces was found: when the lipid headgroup was changed from zwitterionic to negatively charged, membrane association of the peptide became completely entropy-driven.     

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.     

A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis.

Previous studies (D.L. Sawicki, D. B. Barkhimer, S. G. Sawicki, C. M. Rice, and S. Schlesinger, Virology 174:43-52, 1990) identified a temperature-sensitive (ts) defect in Sindbis virus nonstructural protein 4 (nsP4) that reactivated negative-strand synthesis after its normal cessation at the end of the early phase of replication. We now report identification of two different ts alterations in nsP2 of Ala-517 to Thr in ts17 or Asn-700 to Lys in ts133 that also reactivated negative-strand synthesis. These same mutations caused severely reduced protease processing by nsP2 and recognition of the internal promoter for subgenomic mRNA synthesis and were responsible for the conditional lethality and RNA negativity of these mutants. Reactivation of negative-strand synthesis by mutations in nsP2 resembled that in nsP4: it was a reversible property of stable replication complexes and did not require continuation of viral protein synthesis. Recombinant viruses expressing both mutant nsP2 and nsP4 reactivated negative-strand synthesis more efficiently than did either mutant protein alone, consistent with the hypothesis that both nsP2 and nsP4 participate in template recognition. We propose that these alterations cause nsP2 and nsP4 to switch from their normal preference to recognize negative strands as templates to recognize positive strands and thereby mimic the initial formation of a replication complex.     

Assembly of two independent populations of flock house virus particles with distinct RNA packaging characteristics in the same cell

Flock House virus (FHV; Nodaviridae) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.     

Sendai virus vectors as an emerging negative-strand RNA viral vector system

The power to manipulate the genome of negative-strand RNA viruses, including the insertion of additional non-viral genes, has led to the development of a new class of viral vectors for gene transfer approaches. The murine parainfluenza virus type I, or Sendai virus (SeV), has emerged as a prototype virus of this vector group, being employed in numerous in vitro as well as animal studies over the last few years. Extraordinary features of SeV are the remarkably brief contact time that is necessary for cellular uptake, a strong but adjustable expression of foreign genes, efficient infection in the respiratory tract despite a mucus layer, transduction of target cells being independent of the cell cycle, and an exclusively cytoplasmic replication cycle without any risk of chromosomal integration. In this review we describe the current knowledge of Sendai virus vector (SeVV) development as well as the results of first-generation vector applications under both in vitro and in vivo conditions. So far, Sendai virus vectors have been identified to be a highly efficient transduction tool for a broad range of different tissues and applications. Future directions in vector design and development are discussed.