Paul Kaesberg

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 picornaviruses1–4, it is involved in the creation of defective interfering (DI) RNAs of positive- and negative-strand viruses5–9 and is implicated in the synthesis of the messenger RNAs of influenza virus10 and coronavirus11. 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.

Complete nucleotide sequence of brome mosaic virus RNA3

Abstract A complementary DNA copy of the 3a cistron of dicistronic RNA3 from brome mosaic virus has been cloned and sequenced. Based on this and other data, the complete nucleotide sequence of brome mosaic virus RNA3 is presented. Following a 91 base 5′ non-coding region, the 909 base 3a cistron encodes a protein of predicted molecular weight 32,480. Between the 3a and coat protein cistrons is an approximately 250 base intercistronic non-coding region. Overlapping the intercistronic region by 9 bases, the last 876 bases of RNA3 encode the subgenomic coat protein messenger, RNA4, completely and without interruption. A third long open reading frame of possible evolutionary significance overlaps the 3a and coat protein cistrons. An internal poly(A) of heterogeneous length (16 to 22 bases) occurs in the intercistronic region just 20 bases 5′ to the start of RNA4 sequences. Averaging the poly(A) heterogeneity, the length of brome mosaic virus RNA3 is 2114 bases.

A pH-Induced Structural Change in Bromegrass Mosaic Virus

Bromegrass mosaic virus undergoes a reversible decrease in its sedimentation coefficient when the pH is raised above pH 6.7. At pH 6 the sedimentation coefficient is 87 S, at pH 7 it is 79 S. Intrinsic viscosities determined at pH 6 and 7 are 3.64 and 5.5 x 10(-2) dl/gm. Diffusion coefficients are 1.56 x 10(-7) cm(2)/sec. and 1.44 x 10(-7) cm(2)/sec., respectively. Radii of gyration, measured by x-ray scattering, are 106 and 128 A. However, appropriate combination of sedimentation, diffusion, and viscosity coefficients at pH 6 and 7 yield the same molecular weight. Also, the zero-angle value of x-ray-scattered intensity, which is a function of molecular weight, is the same at the two pHs. These results suggest that bromegrass mosaic virus particles undergo a pH-induced change in structure. This change causes, among other things, an increase in the susceptibility of the particles to degradation by pancreatic ribonuclease. The shape of the titration curve between pH 6.3 and 6.9 is anomalous.

The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 x 10(6) was calculated from the sedimentation coefficient, S degrees (20,w) = 86.2S, the diffusion coefficient, D(20,w) = 1.55 x 10(-7) cm(2)/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 x 10(6) atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.

Structure of the black beetle virus genome and its functional implications

Abstract 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.

Isolation and properties of RNA from bromegrass mosaic virus

RNA from bromegrass mosaic virus was isolated by treatment of the virus with phenol in the presence of bentonite and sodium dodecyl sulfate. The RNA sedimented as three distinct components in the ultracentrifuge. Their sedimentation coefficients, S20,w0, in 10−1 M -KC1 + 10−2 M -potassium acetate + 10−3 M -MgCl2 + 10−3 M -CaCl2 (pH 5·5), were 14·0 s, 22·3 s and 26·8 s. Essentially no change in the sedimentation coefficients of the three components or in their relative amounts was observed after incubation at 20°C for 48 hours or after solution in and recovery from formamide. The identical three-component sedimentation pattern was found with RNA isolated by treatment of the virus with 1 M -CaCl2. RNA isolated from virus variously grown, harvested, purified and treated showed the same pattern. Addition of partially purified host-plant nuclease to the RNA resulted in comparable degradation of each component rather than conversion of the large component into the two smaller components. It was concluded that the three components of bromegrass mosaic virus RNA were not artifacts of virus or RNA isolation but represented the situation in the virus particles themselves in the infected host. The components were separated by sucrose density-gradient centrifugation. Their molecular weights, calculated from the Scheraga-Mandelkern equation, were 3 × 105, 7 × 105 and 1·0 × 106. Base compositions of the three components were similar. It is believed that some bromegrass mosaic virus particles contain the large component whereas others contain the two smaller components. Possibly the two smaller components are pieces of the large component resulting from a single cleavage at some stage in virus synthesis.

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.

Translation of the RNAs of brome mosaic virus: the monocistronic nature of RNA1 and RNA2.

Abstract Each of the two largest brome mosaic virus RNAs, RNA1 and RNA2, directs the synthesis of a large protein in cell-free extracts derived from wheat embryo. The size of each protein represents the translation of almost the entire length of the corresponding RNA. It was shown previously that brome mosaic virus RNA4 directs the synthesis of the coat protein and that brome mosaic virus RNA3, although it also contains the coat protein cistron, is translated mostly into a single product unrelated to the coat protein (Shih & Kaesberg, 1973). Thus, the brome mosaic virus genome encodes a total of four proteins.

Small-angle X-ray scattering from turnip yellow mosaic virus☆

Abstract The small-angle X-ray scattering has been measured from solutions of turnip yellow mosaic virus and the associated protein. The data indicate that both particles are near spherical and about 140 A in radius. The virus is approximately uniform electron density. The protein is a water-filled spherical shell with a ratio of inner to outer radius of 0.75. The 140 A quoted represents the radius of the protein structure excluding external hydration. Thus both particles have large internal hydrations. A reasonable assignment of hydration to the protein and nucleic acid fractions of the virus particle is possible, which gives the particle an approximately uniform electron density.

Cleavage of the N-terminal formymlethionine residue from a bacteriophage coat protein in vitro☆

Abstract Studies were made of the N-terminal formylmethionine content of nascent and complete coat protein of bacteriophage Qβ synthesized in an Escherichia coli cell-free system. Under normal conditions of cell-free protein synthesis the formylmethionine residue was retained by all the nascent chains but by only about 50% of the completed coat protein molecules. If 2-mercaptoethanol was omitted from the cell-free system, the formylmethionine residue was cleaved during the course of peptide chain elongation. All nascent peptides which contained fewer than 40±5 amino acids retained the formylmethionine residue. Thereafter, the proportion of nascent peptides lacking the residue increased with peptide length to about 70% for nearly full length nascent peptides and complete released coat protein molecules.