Protein-protein interactions and glycerophospholipids in bromovirus and nodavirus RNA replication.

The plant bromoviruses and animal nodaviruses are distinct groups of positive strand RNA viruses that have proven to be useful models for RNA replication studies. Bromoviruses encode two large proteins required for RNA replication: 1a contains domains implicated in helicase and capping functions, and 2a contains a central polymerase-like domain. Using immunoprecipitation and far-western blotting, we have now shown that 1a and 2a form a specific complex in vitro and have mapped the interacting domains. Molecular genetic data implicate the 1a-2a complex in RNA replication and suggest that it supports coordinate action of the putative helicase, polymerase, and capping domains. The locations of the interacting 1a and 2a domains have implications for replication models and the evolution of virus genomes bearing homologous replication genes in fused vs. divided forms. For the nodavirus Flock house virus (FHV), a true RNA replicase has been isolated that carries out complete, highly active replication of added FHV RNA, producing newly synthesized positive strand RNA in predominantly ssRNA form. Positive strand RNA synthesis in this FHV cell-free system is strongly dependent on the addition of any of several glycerophospholipids. Positive strand RNA synthesis depends on the complete glycerophospholipid structure, including the polar head group and diacyl glycerol lipid portion, and is strongly influenced by acyl chain length.

Active complete in vitro replication of nodavirus RNA requires glycerophospholipid.

Flockhouse virus (FHV) is a member of the nodavirus group of positive-strand RNA viruses. In the absence of additional compounds, a template-dependent RNA-dependent RNA polymerase extracted from FHV-infected cells synthesizes complementary (-)-strand copies of added FHV RNA to yield a double-stranded RNA product. Upon addition of glycerophospholipid (GPL), this system reproducibly carries out complete highly active replication of added FHV RNA, producing newly synthesized (+)-strand RNA in predominantly single-stranded RNA form. This accounts for previously observed effects of Lipofectin (a mixture of GPL and cationic lipid) in the system. All tested neutral and negatively charged GPLs except phosphatidic acid support complete FHV RNA replication in this in vitro system, as do phospholipid extracts from uninfected and FHV-infected cells. Neither sphingomyelin, a membrane phospholipid that is not derived from glycerol, nor cholesterol supported FHV RNA replication. Testing of compounds derived from GPL shows that the ability of active GPL to support FHV (+)-strand RNA synthesis is dependent on the structures of both the head group and the acyl chains. Neither the phosphorylated head group nor the diacylglycerol lipid moiety alone supports RNA replication. The length and saturation of acyl chains strongly influence the ability of GPL to support RNA replication. Other characteristics of this in vitro RNA replication system and the possible role played by membranes and their components in FHV RNA replication are discussed.

Effects of sized heparin oligosaccharide on the interactions of Chinese hamster ovary cell with thrombospondin.

Binding and degradation of TSP by CHO cells and adhesion of CHO cells to substrate-adsorbed TSP are mediated by cell surface PGs and inhibitable by heparin. In order to learn how these three processes are related, we studied the effects of defined heparin oligosaccharides up to 18-mer produced by nitrous acid digestion. There was a complex correlation among oligosaccharide chain length, affinity of oligosaccharide for TSP in a solid phase binding assay, and potencies of oligosaccharide in inhibition of the three cellular processes. Inhibition of degradation was more sensitive to shorter oligosaccharides than inhibition of binding. For instance, the 10-mer inhibited binding of TSP to cells by 10% and degradation by 70%. Punctate immunofluorescence of cell surface bound TSP was replaced by a diffuse pattern after incubation in the presence of the 10-mer. These results suggest that the clustering of TSP on the cell surface may trigger endocytosis and degradation. Inhibition of binding of TSP to cells, in turn, was more sensitive to midsized oligosaccharides than inhibition of cell adhesion to adsorbed TSP. Inhibition of adhesion correlated with the ability of oligosaccharides to block binding of 125I-heparin to adsorbed TSP.

Biology of cancer and aging.

The greatest risk factor for cancer is aging. Human cancer incidence increases exponentially with advancing age. Cancer growth rate and potential for metastatic spread may be influenced by age-specific change in host response. Because cancer and aging are, thus, inextricably linked, the American Cancer Society should encourage submission of research proposals that address the mechanisms of aging and how aging alters cancer development.

Synthesis of template-sense, single-strand Flockhouse virus RNA in a cell-free replication system.

Cell-free extracts of Drosophila melanogaster cells infected with FHV (Flockhouse virus) have the capacity to synthesize FHV RNA. When such extracts are incubated appropriately, ss and ds FHV RNA 1, -2, and -3 can be detected as synthesis products. Pulse-chase analyses indicate that radioactivity, incorporated into ds RNA 1, -2, and -3 is chased into ss RNA 1, -2, and -3, suggesting that these ds RNAs are intermediates in FHV RNA replication. When the cell-free extracts are treated with detergents to solubilize cellular membranes, only ds RNA 1, -2, and -3 can be detected as synthesis products. The cell-free extracts, treated with a ribonuclease to remove endogenous templates, have the capacity to replicate FHV RNA 1 and -2 when these RNAs are provided as templates, together with lipofectin. Both ss and ds RNA 1 and -2 are detected as synthesis products. When RNA3 is provided as a template, only ds RNA3 is synthesized. In the absence of lipofectin or in the presence of detergent, only ds RNAs are detected as synthesis products and all the incorporated radioactivity is found in the strand complementary to that of the template. Our results are consistent with a mechanism in which second-strand synthesis, although not complementary strand synthesis, requires intact membranes. They also suggest that ds RNA3 is an intermediate in the synthesis of ss RNA3.

Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography.

The genomic RNA2s of nodaviruses encode a single gene, that of protein alpha, the precursor of virion proteins beta and gamma. We compared the sequences of the RNA2s of the nodaviruses, black beetle virus (BBV), flock house virus, boolarra virus and nodamura virus, with the objective of identifying homologies in the primary and secondary structure of these RNAs and in the structure of their encoded protein. The sequences of the four RNAs were found to be similar, so that homologous regions relating to translation and RNA replication were readily identified. However, the overall, secondary structures in solution, deduced from calculations of optimal Watson-Crick base-pairing configurations, were very different for the four RNAs. We conclude that a particular, overall, secondary structure in solution within host cells is not required for virus viability. The partially refined X-ray structure of BBV (R = 26.4% for the current model) was used as a framework for comparing the structure of the encoded proteins of the four viruses. Mapping of the four protein sequences onto the BBV capsid showed many amino acid differences on the outer surface, indicating that the exteriors of the four virions are substantially different. Mapping in the beta-barrel region showed an intermediate level of differences, indicating that some freedom in choice of amino acid residues is possible there although the basic framework of the capsids is evidently conserved. Mapping onto the interior surface of the BBV capsid showed a high degree of conservation of amino acid residues, particularly near the protein cleavage site, implying that that region is nearly identical in all four virions and has an essential role in virion maturation, and also suggests that all four capsid interior surfaces have similar surfaces exposed to the viral RNA. Apart from a small portion of the C promoter, the amino terminus of the BBV protein (residues 1 to 60) is crystallographically disordered and the amino acid residues in that region are not well conserved. The disordered portion of the BBV protein clearly projects from the capsid inner surface into the interior of the virion, the region occupied by the viral RNA. In all four viruses, residues 1 to 60 had a high proportion of basic residues, suggesting a virus-specific interaction of the amino terminus with the virion RNA.

Genomic RNA of an insect virus directs synthesis of infectious virions in plants.

Newly synthesized virions of flock house virus (FHV), an insect nodavirus, were detected in plant cells inoculated with FHV RNA. FHV was found in whole plants of barley (Hordeum vulgare), cowpea (Vigna sinensis), chenopodium (Chenopodium hybridum), tobacco (Nicotiana tabacum), and Nicotiana benthamiana and in protoplasts derived from barley leaves. Virions produced in plants contained newly synthesized RNA as well as newly synthesized capsid protein. These results show that the intracellular environment in these plants is suitable for synthesis of a virus normally indigenous only to insects. Such synthesis involves, minimally, translation of viral RNA, RNA replication, and virion assembly. Inoculation of barley protoplasts with FHV virions resulted in synthesis of small amounts of progeny virions, suggesting that FHV virions are capable of releasing their RNA in plant cells. In N. benthamiana, virions resulting from inoculation with RNA were detected not only in inoculated leaves but also in other leaves of inoculated plants, suggesting that virions could move in this plant species. Such movement probably occurs by a passive transport through the vascular system rather than by an active transport involving mechanisms that have evolved for plant viruses.

The importance of immunesenescence in the incidence and malignant properties of cancer in hosts of advanced age.

Profound changes in the biology of cancer occur as people and experimental animals age. Many naturally occurring tumors in humans and experimental animals show slower growth with advancing age and longer host survival. The most likely cause for this alteration in tumor biology is the universal phenomenon of cellular immunesenescence. At the onset of sexual maturation, the thymus begins to involute. With this involution, a progressive decline is observed in T-cell functions, e.g., lymphokine production, response to mitogens, and response to antigenic stimulation. It has been proposed that the immune system plays an important role in the stimulation of tumor growth. This role in poorly antigenic tumors may overshadow the role of the immune system as a tumor suppressor. It has been shown that poorly antigenic murine tumors grow more slowly in immune deficient or aged mice. Human tumors are generally poorly antigenic and many (such as lung, breast, and colon carcinomas) also show decreased growth rates in older adults. This article describes age-related changes in the immune system and discusses the theories of immune enhancement of tumor growth. Consideration is also given to the explanation of increased incidence of cancer in elderly patients and the potential role of the immune system in this phenomenon.

Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparan sulfate proteoglycan.

Thrombospondin is a 420-kD platelet alpha-granule glycoprotein that binds specifically to heparin. We examined adhesion to thrombospondin of CHO K1 cells and three mutant CHO lines with varying deficiencies in glycosaminoglycan (GAG) synthesis. In an experiment in which the parent line (K1) had 78% adherence to thrombospondin adsorbed to tissue culture plastic, CHO S745 cells, with less than 6% normal GAG synthesis had 11% adherence. CHO S677 cells, with decreased heparan sulfate proteoglycan but increased chondroitin sulfate proteoglycan, had 42% adherence. CHO S803 cells, with decreased heparan sulfate proteoglycan and normal chondroitin sulfate proteoglycan, had 31% adherence. Heparin inhibited K1 cell adhesion to thrombospondin, but not fibronectin, in a concentration-dependent manner. Dermatan sulfate but not chondroitin sulfate was also inhibitory. There was markedly decreased K1 cell adhesion to a thrombospondin core fragment that lacked the heparin binding NH2-terminal domain. Purified heparin binding domain, although poorly adhesive when adsorbed to substratum, inhibited cell adhesion to intact thrombospondin. Adhesion was better for all cell lines tested, including three human tumor cell lines, when thrombospondin was adsorbed at pH 4.0 compared with pH 7.4. When adsorption of thrombospondin was done at pH 7.4, cell adhesion was better when thrombospondin was adsorbed in the presence of greater than or equal to 0.6 mM calcium, compared to 0.1 mM calcium or EDTA. These findings suggest that thrombospondin can adsorb to plastic with varying degrees of exposure of a cell adhesion domain. We conclude that the thrombospondin cell adhesion receptor on CHO cells is a heparan sulfate proteoglycan, and that cell adhesion to thrombospondin depends on conformation of adsorbed thrombospondin.

Infectious positive- and negative-strand transcript RNAs from bacteriophage Q beta cDNA clones.

Plasmids containing full-length cDNA copies of the Q beta RNA phage genome and flanking T7 promoters were constructed. Positive-strand Q beta RNA, generated by in vitro transcription of these plasmids with T7 RNA polymerase, was infectious to Escherichia coli spheroplasts. The Q beta replicase gene from the cloned DNA was subcloned and expressed in E. coli cells by means of a thermoinducible plasmid. Full-length, negative-strand Q beta transcripts were infectious when transfected into spheroplasts containing the induced replicase gene.

Concurrent development of a thymic cyst and mediastinal Hodgkin's disease.

A case is presented of a young adult female who presented with nodular sclerosing Hodgkin's disease and a mediastinal mass that proved to be a thymic cyst. Prior to this illness, a chest x-ray had been obtained that did not show a mediastinal mass. This case illustrates that thymic cysts may arise simultaneously with mediastinal Hodgkin's disease, rather than as a result of treatment or being present congenitally.

Sequence and organization of southern bean mosaic virus genomic RNA.

The genomic RNA sequence of the cowpea strain of southern bean mosaic virus (SBMV-C) has been determined. The genome is 4194 nucleotides in length and has four open reading frames. A 5' proximal open frame, from base 49 to base 603, corresponds to the length of the P4 proteins translated in cell-free extracts from full-length and smaller virion RNA. The largest open frame extends from base 570 to base 3437 and encodes the two largest proteins translated in cell-free extracts from full-length virion RNA. Segments of this open reading frame's predicted amino acid sequence resemble those of known viral RNA polymerases, ATP-binding proteins, and viral genome-linked proteins. A third open frame extends from base 1895 to base 2380 and has not been correlated with an in vitro translation product. The fourth open reading frame is located in the 3' terminal region of the genome extending from base 3217 to base 4053. This frame encodes the SBMV capsid protein which is translated from subgenomic, virion RNA.

Modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3' end.

The genome of brome mosaic virus (BMV) is comprised of three (+) strand RNAs, each containing a similar, highly structured, 200 base long sequence at its 3' end. A 134 base subset of this sequence contains signals directing interaction of the viral RNA with BMV RNA replicase, ATP,CTP:tRNA nucleotidyl transferase and aminoacyl tRNA synthetase. A series of mutants containing deletions within this region, previously constructed and tested in vitro for the effect on replication and aminoacylation activities, has now been assayed in vitro for adenylation function and in vivo for ability to replicate in isolated protoplasts and whole plants. These tests indicate that features of viral RNA recognized by BMV replicase overlap those directing adenylation, but are distinct from those directing aminoacylation. Consequently, the lethality of a deletion preferentially inhibiting aminoacylation suggests that this function may have an essential role contributing to viral replication in vivo. An RNA3 mutant bearing a 20-base deletion yielding normal levels of aminoacylation and enhanced levels of replicase template activity and adenylation in vitro was able to replicate in protoplasts and plants; however, its accumulation in protoplasts was reduced relative to wild-type. This suggests that additional functions affecting the replication and accumulation of viral RNA reside in the conserved 3' sequence.

Genetic recombination between RNA components of a multipartite plant virus.

Genetic recombination of DNA is one of the fundamental mechanisms underlying the evolution of DNA-based organisms and results in their diversity and adaptability. The importance of the role of recombination is far less evident for the RNA-based genomes that occur in most plant viruses and in many animal viruses. RNA recombination has been shown to promote the evolutionary variation of picornaviruses, it is involved in the creation of defective interfering (DI) RNAs of positive- and negative-strand viruses and is implicated in the synthesis of the messenger RNAs of influenza virus and coronavirus. However, RNA recombination has not been found to date in viruses that infect plants. In fact, the lack of DI RNAs and the inability to demonstrate recombination in mixedly infected plants has been regarded as evidence that plants do not support recombination of viral RNAs. Here we provide the first molecular evidence for recombination of plant viral RNA. For brome mosaic virus (BMV), a plus-stranded, tripartite-genome virus of monocots, we show that a deletion in the 3' end region of a single BMV RNA genomic component can be repaired during the development of infection by recombination with the homologous region of either of the two remaining wild-type BMV RNA components. This result clearly shows that plant viruses have available powerful recombinatory mechanisms that previously were thought to exist only in animal hosts, thus they are able to adapt and diversify in a manner comparable to animal viruses. Moreover, our observation suggests an increased versatility of viruses for use as vectors in introducing new genes into plants.

Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus.

RNA transcripts of cloned cDNA of the genomic RNAs of BBV (black beetle virus) are infectious to cultured cells of Drosophila melanogaster. Individual transcripts had approximately 10% of the infectivity of the corresponding authentic virion RNA. Progeny virus resulting from transcript infection was phenotypically indistinguishable from the progenitor virus used to generate the original cDNA forms as judged by sucrose density gradient sedimentation, specific infectivity, plaque morphology, and serology. Although the transcript RNAs used to produce this virus had 20 nonviral bases headed by a capping group at their 5' termini, these 20 bases were absent in the progeny viral RNAs. The cDNA forms, and therefore the resulting transcript RNAs, should be readily modifiable by the techniques of recombinant DNA technology both for viral studies and for the insertion of foreign genes into the viral genome and thus into the host cytoplasm.

Template-dependent RNA polymerase from black beetle virus-infected Drosophila melanogaster cells.

Infection of cultured cells of Drosophila melanogaster with black beetle virus (BBV) induces an RNA polymerase that is bound to cellular particulate material in a complex with a template RNA. We have solubilized the polymerase by treatment of the relevant particulates with detergents such as dodecyl-beta-D-maltoside. The polymerase activity was made dependent upon exogenous RNA by destruction of the endogenous template RNA with micrococcal nuclease. Addition of BBV RNA1 or RNA2 induced synthesis of full-length negative-strand RNA isolated as a double-stranded complex with the added RNA. Newly synthesized plus strands were also detected in the RNA2 complexes. Certain other viral RNAs also induced synthesis of their negative strands.

Structure of the black beetle virus genome and its functional implications.

The black beetle virus (BBV) is an isometric insect virus whose genome consists of two messenger-active RNA molecules encapsidated in a single virion. The nucleotide sequence of BBV RNA1 (3105 bases) has been determined, and this, together with the sequence of BBV RNA2 (1399 bases) provides the complete primary structure of the BBV genome. The RNA1 sequence encompasses a 5' non-coding region of 38 nucleotides, a coding region for a protein of predicted molecular weight 101,873 (protein A, implicated in viral RNA synthesis) and a 3' proximal region encoding RNA3 (389 bases), a subgenomic messenger RNA made in infected cells but not encapsidated into virions. The RNA3 sequence starts 16 bases inside the coding region of protein A and contains two overlapping open reading frames for proteins of molecular weight 10,760 and 11,633, one of which is believed to be protein B, made in BBV-infected cells. A limited homology exists between the sequences of RNA1 and RNA2. Sequence regions have been identified that provide energetically favorable bonding between RNA2 and RNA1 possibly to facilitate their common encapsidation, and between RNA2 and negative strand RNA1 possibly to regulate the production of RNA3.

Sequence of the black beetle virus subgenomic RNA and its location in the viral genome.

BBV (black beetle virus) RNA3, the subgenomic messenger RNA for BBV protein B and its double-stranded form (dsRNA3) were purified from cells infected with BBV and were sequenced. RNA3 is 389 bases long. The sequence is homologous to that of the 3'-terminal region of virion RNA1. RNA3 has a very limited homology to virion RNA2. RNA3 is capped at its 5' terminus and has a structural feature at its 3' terminus that renders it inert to the action of the enzymes RNA ligase and poly(A) polymerase. RNA3 has two overlapping reading frames for putative proteins of size 10,768 and 11,633 Da. The positive and negative strands of dsRNA3 are not capped and correspond in length and sequence to RNA3 itself.