Filamentous phage assembly: morphogenetically defective mutants that do not kill the host

The filamentous phage virion is assembled without killing the host, by extrusion of the DNA through the envelope and concomitant acquisition of coat proteins from the inner membrane. When assembly is blocked, however, intracellular phage DNA and gene products accumulate and the host is killed. This "cell killing" is largely absent in phage fd-tet, which carries a tetracycline-resistance determinant within the origin of minus-strand synthesis; as a result of the replication defect, phage DNA does not accumulate to high levels intracellularly when virion assembly is blocked. This allows morphogenetically defective mutants except those ablating gene V to be freely propagated in tetracycline-containing medium and studied in the absence of the confounding factor of cell morbidity. Because cultures can be initiated by transfection in the complete absence of input virions, extremely low levels of phage production can be assayed. Using this system, I show that genes III, VI, I, and IV are not required to form the complex between viral DNA and gene-V protein that is the intracellular precursor to mature virions; that genes I and/or IV are absolutely (or nearly absolutely) required for assembly; and that mos, a cis-acting sequence previously shown to enhance phage yield in some circumstances, is without such effect in others.     

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.     

Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly

Assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. Closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. The Closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvement of gene products of Citrus tristeza virus (CTV) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins were required for efficient virion assembly. Competency of virion assembly of different CTV mutants was assayed by their ability to be serially passaged in Nicotiana benthamiana protoplasts using crude sap as inoculum, and complete and partial virus particles were analyzed by serologically specific electron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p20, and p23 genes were not needed for virion formation. However, deletion of either minor- or major-coat protein resulted in formation of short particles which failed to be serially transferred in protoplasts, suggesting that both coat proteins are required for efficient virion assembly. Deletion or mutation of HSP70h and/or p61 dramatically reduced passage and formation of full-length virions. Frameshift mutations suggested that the HSP70h and p61 proteins, not the RNA sequences, were needed for virion assembly. Substitution of the key amino acid residues in the ATPase domain of HSP70h, Asp(7) to Lys or Glu(180) to Arg, reduced assembly, suggesting that the chaperone-like ATPase activity is involved in assembly. Both HSP70h and p61 proteins appeared to contribute equally to assembly, consistent with coordinate functions of these proteins in closterovirus virion formation. The requirement of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions.     

Assembly of nucleocapsids with cytosolic and membrane-derived matrix proteins of vesicular stomatitis virus.

During budding of vesicular stomatitis virus (VSV), the viral matrix (M) protein binds the viral nucleocapsid to the host plasma membrane and condenses the nucleocapsid into the tightly coiled nucleocapsid-M protein (NCM) complex observed in virions. In infected cells, the viral M protein exists mostly as a soluble molecule in the cytoplasm, and a small amount is bound to the plasma membrane. Despite the high concentrations of M protein and intracellular nucleocapsids in the cytoplasm, they are not associated with each other except at the sites of budding. The experiments presented here address the question of why M protein and nucleocapsids associate with each other only at the plasma membrane but not in the cytoplasm of infected cells. An assay for exchange of soluble M protein into NCM complexes in vitro was used to show that both cytosolic and membrane-derived M proteins bound to virion NCM complexes with affinities similar to that observed for virion M protein, indicating that both cytosolic and membrane-derived M proteins are competent for virus assembly. However, neither cytosolic nor membrane-derived M protein bound to intracellular nucleocapsids with the same high affinity observed for virion NCM complexes. Cytosolic M protein was able to bind intracellular nucleocapsids, but with an affinity approximately eightfold less than that observed in virion NCM complexes. Membrane-derived M protein exhibited little or no binding activity for intracellular nucleocapsids. These data indicate that intracellular nucleocapsids, and not intracellular M proteins, need to undergo an assembly-initiating event in order to assemble into an NCM complex. Since neither membrane-derived nor cytosolic M protein could initiate high-affinity binding to intracellular nucleocapsids, the results suggest that another viral or host factor is required for assembly of the NCM complex observed in virions.     

Adenovirus DNA synthesis is coupled to virus assembly

The relationship between viral DNA synthesis and virion assembly was studied in adenovirus type 2 infected HEp2 cells. When cells were infected at the restrictive temperature with ts3, an assembly-negative mutant which permits normal viral DNA and protein synthesis, labeled and shifted to the permissive temperature, only de novo synthesized nonradioactive viral DNA was encapsidated. This suggested that only concurrently synthesized DNA is encapsidated. Blocking protein or DNA synthesis with cycloheximide or hydroxyurea after the temperature shift inhibited virus assembly. Therefore efficient virus assembly requires both concurrent protein and DNA synthesis. When DNA synthesis was arrested by shifting ts125 infected cells to the restrictive temperature, protein synthesis continued but assembly was completely blocked. Sucrose gradient sedimentation analysis of nuclear extracts of wt and ts3 infected cells provided evidence in support of a physical coupling between replication complexes and virus assembly complexes. Further evidence of coupling was also shown by preferential pulse labeling of the molecular right end of the genome isolated from reversibly cross-linked assembly intermediate particles. While DNA replication is not dependent on concurrent virion assembly, at least some significant proportion of replication complexes appear to be coupled to and are prerequisite for virion assembly.     

Structural evolution of the P22-like phages: comparison of Sf6 and P22 procapsid and virion architectures

Coat proteins of tailed, dsDNA phages and in herpesviruses include a conserved core similar to the bacteriophage HK97 subunit. This core is often embellished with other domains such as the telokin Ig-like domain of phage P22. Eighty-six P22-like phages and prophages with sequenced genomes share a similar set of virion assembly genes and, based on comparisons of twelve viral assembly proteins (structural and assembly/packaging chaperones), these phages are classified into three groups (P22-like, Sf6-like, and CUS-3-like). We used cryo-electron microscopy and 3D image reconstruction to determine the structures of Sf6 procapsids and virions (~ 7 Å resolution), and the structure of the entire, asymmetric Sf6 virion (16-Å resolution). The Sf6 coat protein is similar to that of P22 yet it has differences in the telokin domain and in its overall quaternary organization. Thermal stability and agarose gel experiments show that Sf6 virions are slightly less stable than those of P22. Finally, bacterial host outer membrane proteins A and C were identified in lipid vesicles that co-purify with Sf6 particles, but are not components of the capsid.     

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.     

Specific in vitro association between the hepatitis C viral genome and core protein.

Little is known about the molecular interactions required for hepatitis C virion assembly. The 5' noncoding region (5'NCR) of the RNA genome is highly conserved and has extensive secondary structure. The highly basic core protein is rich in arginine and lysine residues. We postulate that a specific interaction between these structures may be important for virion assembly. Using an RNA gel mobility shift assay, a specific interaction has been demonstrated between the RNA of the 5'NCR and recombinant core protein. Proteins from other regions of the virus do not interact with the viral RNA. The interaction is inhibited competitively by unlabelled sense polarity RNA, but antisense 5'NCR RNA and nonspecific RNAs compete only at much higher concentrations. These data suggest that there is a specific interaction between the 5'NCR of the hepatitis C virus (HCV) genome and HCV core protein. This interaction may be important for the specific encapsidation of the viral genome during HCV replication.     

Morphogenesis of the tail of bacteriophage lambda I. In vitro intratail complementation

By studying nonsense mutants in ten of the 11 known tail genes of bacteriophage lambda we obtained the following results. Concentrated tail(-)-lysates complement each other in nearly all possible (45) combinations. The efficiency of complementation is very dependent on physical parameters (lysis procedure, concentration, temperature) and on the mutants used for complementation (i.e., type of precursors present, absence or presence of abortive structures, polarity effects of some nonsense mutants). Gene product dosage experiments, in which the ratio of concentrations of the different precursors present in different lysates is varied up to four orders of magnitude, allow us to distinguish five classes of intratail complementations. By these studies we detected that the morphogenetic action of at least two gene products, pV and pM, is extremely concentration-dependent. Furthermore the gene product dosage experiments allow us to deduce for five gene products the following sequence of morphogenetic action during lambda tail assembly: (pJ,pM), (pV,pU), pZ. Finally, we find that our in vitro complementation data are consistent with already known polarity properties of nonsense tail(-)-mutants of phage lambda.     

Evolution of mosaically related tailed bacteriophage genomes seen through the lens of phage P22 virion assembly

The mosaic composition of the genomes of dsDNA tailed bacteriophages (Caudovirales) is well known. Observations of this mosaicism have generally come from comparisons of small numbers of often rather distantly related phages, and little is known about the frequency or detailed nature of the processes that generate this kind of diversity. Here we review and examine the mosaicism within fifty-seven clusters of virion assembly genes from bacteriophage P22 and its “close” relatives. We compare these orthologous gene clusters, discuss their surprising diversity and document horizontal exchange of genetic information between subgroups of the P22-like phages as well as between these phages and other phage types. We also point out apparent restrictions in the locations of mosaic sequence boundaries in this gene cluster. The relatively large sample size and the fact that phage P22 virion structure and assembly are exceptionally well understood make the conclusions especially informative and convincing.     

Recruitment of host translation initiation factor eIF4G by the Vaccinia Virus ssDNA-binding protein I3

Poxviruses are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells within discrete compartments termed viral factories. Recent work has shown that the prototypical poxvirus, Vaccinia Virus (VacV) sequesters components of the eukaryotic translation initiation complex eIF4F within viral factories while also stimulating formation of eIF4F complexes. However, the forces that govern these events remain unknown. Here, we show that maximal eIF4F formation requires viral DNA replication and the formation of viral factories, suggesting that sequestration functions to promote eIF4F assembly, and identify the ssDNA-binding protein, I3 as a viral factor that interacts and co-localizes with the eIF4F scaffold protein, eIF4G. Although it did not adversely affect host or viral protein synthesis, I3 specifically mediated the binding of eIF4G to ssDNA. Combined, our findings offer an explanation for the specific pattern and temporal process of eIF4G redistribution and eIF4F complex assembly within VacV-infected cells.     

The polar alkaline disassembly of papaya mosaic virus

The disassembly of papaya mosaic virus (PMV) at alkaline pH (pH 10) was monitored by following the decrease of turbidity at 320 nm and by electron microscopy. Most of the virion particle population undergoes a sequential loss of protein subunits starting exclusively from the 3'OH end of the RNA, but a small fraction resists degradation. The progressive decrease in size distribution suggests a sequential process and the presence of "brush-like" structures at one end of the virus particle favors a polar model for disassembly. Furthermore, after treatment with nucleases to digest the exposed RNA tails, the 5' cap structure (m7GpppGp) was found to be conserved in the remaining nucleoprotein intermediates. This indicates that the disassembly of PMV occurs from the 3' terminus to the 5' end, a polarity opposite to that of viral assembly. These RNA fragments (containing the 5' end) retain their ability to reassemble with the viral protein. This result is consistent with earlier evidence that the initiation site for virus assembly is located at the 5' end of PMV-RNA. Electron microscope examination suggests the presence of meta-stable size classes during the disassembly process. This observation might indicate a variability of the RNA-protein interactions along the RNA molecules.     

Functions of baseplate components in bacteriophage T4 infection. I. Dihydrofolate reductase and dihydropteroylhexaglutamate

Phage T4 are known to be inactivated by conjugase, anti-dihydrofolate reductase, and NADPH. We examined the specificity and mechanisms of action of these agents on the normal phage and some adsorption intermediates in order to clarify the stages of infection in which the phage structural components dihydropteroylhexaglutamate and dihydrofolate reductase are involved. Dihydrofolate reductase plays no enzymic role in the virion, but is structurally important during the early stages of phage adsorption. Dihydropteroylhexaglutamate plays a role in the later stages of phage adsorption.     

Self-assembly of double-stranded RNA bacteriophages

Double-stranded RNA viruses infecting bacterial hosts belong to the Cystoviridae family. Bacteriophage phi6 is one of the best characterized dsRNA viruses and shares structural as well as functional similarities with other well-studied eukaryotic dsRNA viruses (e.g. L-A, rotavirus, bluetongue virus, and reovirus). The assembly pathway of the enveloped, triple-layered phi6 virion has been well documented and can be divided into four distinct steps which are (1) procapsid formation, (2) genome encapsidation and replication, (3) nucleocapsid surface shell assembly, and (4) envelope formation. In this review, we focus primarily on the procapsid and nucleocapsid assembly for which in vitro systems have been established. The in vitro assembly systems have been instrumental in revealing assembly intermediates and conformational changes that are common to phi6 and phi8, two cystoviruses with negligible sequence homology. Two viral enzymes, the packaging NTPase (P4) and the RNA-dependent RNA polymerase (P2), were found essential for the nucleation step. The nucleation complex contains one or more tetramers of the major procapsid protein (P1) and is further stabilized by protein P4. Interaction of P1 and P4 during assembly is accompanied by an additional folding of their respective polypeptide chains. The in vitro assembled procapsids were shown to selectively package and replicate the genomic ssRNA. Furthermore, in vitro assembly of infectious nucleocapsids has been achieved in the case of phi6. The in vitro studies indicate that the nucleocapsid coat protein (P8) assembles around the polymerase complex in a template-assisted manner. Implications for the assembly of other dsRNA viruses are also presented.     

Nonsense mutants of the lipid-containing bacteriophage PR4

Thirty-three nonsense mutants of phage PR4 representing 12 complementation groups were isolated. One or two mutants of each group were grown on a suppressor-negative (Su-) host and characterized by the following criteria (i) proteins synthesized, (ii) level of phage DNA synthesis, and (iii) ability to assemble particles. We determined the protein and phospholipid compositions of the particles assembled in an Su- host, the presence of DNA in the particles, and the ability of the particles to adsorb to host cells. Finally each complementation group was tested for the ability to lyse an Su- host. We have identified one protein required for DNA synthesis, five proteins required for proper assembly of the protein coat and lipid membrane of the phage, two proteins required for stable insertion of DNA into the virion, a protein required for adsorption, a protein required for attachment of the adsorption protein to the virion, and a phage-encoded lytic enzyme.     

Use of surface plasmon resonance imaging to study viral RNA: protein interactions

Surface plasmon resonance imaging (SPRi) is an emerging microarray technology that is label-free, rapid and extremely flexible. Here the capabilities of SPRi are demonstrated in results of proof-of-concept experiments detailing a method for studying viral genomic RNA:protein interactions in array format. The principal RNA is the well-characterized origin of assembly (OAS) containing region of Tobacco mosaic virus (TMV) RNA, whereas the principal protein is the primary subunit for TMV virion assembly, the 20S capsid protein aggregate. DNA probes complementary to TMV and non-TMV RNA fragments were covalently attached to a thin gold layer deposited on glass. These DNA probes were used to discreetly capture in vitro transcribed TMV and Red clover necrotic mosaic virus (RCNMV) RNA2 (used as a negative control for the subsequent protein binding). The 4S TMV capsid protein monomers were isolated from TMV particles purified from infected plants of Nicotiana tabacum L. and were induced to form 20S stacked disc aggregates. These 20S stacked disc aggregates were then injected onto the array containing the RNA fragments captured by the DNA probes immobilized on the microarray surface. The discrete and preferential binding of the 20S stacked disc aggregates to the array locations containing the TMV OAS RNA sequence was observed. The results demonstrate that SPRi can be used to monitor binding of large RNA molecules to immobilized DNA capture probes which can then be used to monitor the subsequent binding of complex protein structures to the RNA molecules in a single real-time, label-free microarray experiment. The results further demonstrate that SPRi can distinguish between RNA species that have or do not have an origin of assembly sequence specific for a particular viral capsid protein or protein complex. The broader implications of these results in virology research are found in other systems where the research goals include characterizing the specificity and kinetics of viral or host protein or protein complex interactions with viral nucleic acids.     

Morphogenesis of sandfly viruses (Bunyaviridae family)

The events occurring in the morphogenesis of sandfly fever viruses have been examined by thin-section electron microscopy and by an analysis of the association of virus-specific polypeptides with membranes of infected cells. Two representative sandfly fever viruses have been studied, Karimabad virus (KV) and Punta Toro virus (PTV), which appeared indistinguishable both in terms of virion structure, as monitored by negative staining, and morphogenesis, as observed in thin sections of infected Vero cells. Ammonium molybdate negative staining of purified, glutaraldehyde-fixed virions revealed essentially spherical particles, 87 nm in diameter, in which the surface proteins were constructed into closely packed, hollow, cylindrical subunits measuring 10–11 nm in diameter and 9–10 nm in length. These surface units are located peripherally to a 7-nm membrane bilayer which surrounds a nucleoid of variable electron density. As seen in thin sections of infected cells, the assembly of these particles was first detected at 12 hr after infection, occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Morphologically mature particles were formed by continuous involution (budding) of modified membrane segments into the lumen of these vesicles. Viral ribonucleoprotein (RNP), which was not observed free in the cytoplasm, condensed at the cytoplasmic face of these vesicles at areas at which viral spike structures could be observed at the contralateral (luminal) face. Neither RNP nor spike structures could be observed on adjacent sections of the vesicular membrane, or other membranes, which were not directly involved in the assembly of a budding virion. Analysis of the viral polypeptides in unfractionated membrane vesicles prepared from infected cells demonstrated that KV-specific envelope and nucleocapsid proteins rapidly became membrane bound, whereas a nonstructural polypeptide was found only in cytoplasmic fractions. Chymotrypsin treatment of these vesicles has indicated that at least one viral glycoprotein is inserted into cellular membranes such that approximately 12% of its sequence remains at the cytoplasmic membrane face. Proteolytic removal of this sequence generated an immunoprecipitable, glycosylated fragment which was protected from further proteolysis by its orientation within the vesicle. These morphologic and biochemical data have been used to construct a model for the assembly of these viruses. Virus particles are released from infected cells by exocytosis, a process which does not appear to result in significant modification of cell surface membranes with viral antigens.     

Isolation and characterization of polyoma nucleoprotein complexes

A method for the isolation of polyoma nucleoprotein complexes has been developed using neuraminidase treatment of infected cell lysates. At least three distinct forms of polyoma virion intermediates were identified by their [3H]thymidine labeling kinetics and sedimentation coefficients: a rapidly labeled 95 S "replicating complex" which chases to a 75 S minichromosome and then to a 240 S virion structure. The general properties of these distinct intermediates were similar to those found for SV40. In contrast to SV40, however, a continuum of labeled polyoma viral DNA sedimented between 240 S and 95 S. These complexes were characterized by their release from cell debris with neuraminidase, precipitation with antivirion antibody, complete disruption in 1 M NaCl, and association with hemagglutinating (HA) activity. These intermediates may represent incremental capsid protein additions to the 75 S minichromosome, hypothesized in the current models for SV40 assembly. The ability to isolate a complete complement of polyoma subviral complexes provides a basis for studying the growth defect of polyoma host-range mutants, and the properties of neuraminidase release, hemagglutination, and specific immunoprecipitation suggest purification steps for further characterization of these virion assembly intermediates.