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Background Ponatinib, a third-generation BCR-ABL1 tyrosine kinase inhibitor (TKI), + hyper-CVAD showed remarkable activity against Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and may be superior to chemotherapy + earlier generation TKIs in terms of depth of remission, event-free survival (EFS), and overall survival (OS). However, this regimen's efficacy and tolerability have yet to be externally validated. Here, we summarize our real-world experience with ponatinib + hyper-CVAD for untreated Ph+ ALL and other Ph+ acute or blast phase leukemias. Methods We retrospectively analyzed all adults treated at the University of California, Davis (UCD) from March 2012 to May 2021 with ponatinib + hyper-CVAD upfront. The primary endpoints were 3-year OS and EFS. Secondary endpoints were complete molecular response (CMR), measurable residual disease (MRD) negativity by multiparameter flow cytometry (MFC), complete cytogenetic response (CCyR) rates, and adverse events (AEs). Time to event analyses were done via the Kaplan-Meier method. Patients alive were censored at their last follow-up date. Patients undergoing allogeneic hematopoietic cell transplant (HCT) after 6 months of achieving complete remission (CR) were censored at the time of HCT for the landmark analysis. Patients with missing data were excluded from the response analyses. Results We identified 13 Ph+ ALL patients who received ponatinib + hyper-CVAD for initial induction. The baseline characteristics for the Ph+ ALL patients are summarized in Table 1. The median follow-up was 16 months. The median number of hyper-CVAD cycles completed was 8 (range, 1-8) with ponatinib. Two patients proceeded to HCT in CR1, one at 3.5 months after starting induction, and due to difficulty controlling the patient's concurrent multiple myeloma prior to HCT and recovery from anti-neoplastic therapy, the second was delayed to 46 months after starting induction. The 3-year OS and EFS with ponatinib + hyper-CVAD were each 92% (95% confidence interval, 78.9-100) (Figure 1). Landmark analysis completed 6 months following CR showed a 3-year OS of 100% in patients treated with ponatinib + hyper-CVAD without HCT in first CR (CR1). The CMR, CCyR, and MRD-negativity by MFC rates with ponatinib were all 92.3% (12/13). The median time to CMR, CCyR, and MRD-negativity by MFC were 51 days, 22 days, and 53 days, respectively. Notable AEs with ponatinib include neutropenic fever (92%), bacterial infection (69%), transaminitis (38%), venous thromboembolism (31%), invasive fungal infection (15%), hemorrhage (15%), cerebrovascular accident (CVA) (15%), and tumor lysis syndrome (8%). One patient died during induction with ponatinib due to a bacterial infection. Two patients switched to a different TKI due to a CVA after 4 and 24 months. Only 2 patients did not complete 8 cycles of hyper-CVAD, due to death during induction (n=1) and proceeding to HCT after 3 cycles (n=1). As for similar Ph+ leukemias, 3 chronic myeloid leukemia with lymphoid blast crisis (CML-LBC), 1 CML with mixed phenotype blast crisis (CML-MPBC), and 1 with mixed phenotype acute leukemia (MPAL) were treated with ponatinib + hyper-CVAD. The MPAL patient achieved CMR within 55 days, while the CML-MPBC and 2 CML-LBC patients achieved CMR after HCT. The third CML-LBC patient is in CR with ongoing treatment. After median follow-up of 25 months, all 5 were alive, and only the MPAL patient relapsed 28 months after starting treatment and 1 year after HCT. Conclusion To our knowledge, this is the first report externally validating the efficacy and tolerability of ponatinib + hyper-CVAD for Ph+ ALL. We also show the feasibility of using this regimen in patients with Ph+ CML-LBC, CML-MPBC and MPAL. Despite the small sample size and retrospective nature, our study supports existing data demonstrating that this regimen challenges both the designation of Ph+ ALL as a high-risk disease and the trend to transplant in CR1. Our findings support that ponatinib + hyper-CVAD should be considered a standard of care for Ph+ ALL. Figure 1 Figure 1. Disclosures Kaesberg: Incyte: Speakers Bureau. Rosenberg: Takeda, Janssen: Speakers Bureau. Abedi: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Speakers Bureau; Seattle Genetics: Speakers Bureau. Tuscano: Genentech, Pharamcyclics, Abbvie, BMS, Acrotech, Seattle Genetics, Takeda: Research Funding. Jonas: AbbVie, BMS, Genentech, GlycoMimetics, Jazz, Pfizer, Takeda, Treadwell: Consultancy; 47, AbbVie, Accelerated Medical Diagnostics, Amgen, AROG, Celgene, Daiichi Sankyo, F. Hoffmann-La Roche, Forma, Genentech/Roche, Gilead, GlycoMimetics, Hanmi, Immune-Onc, Incyte, Jazz, Loxo Oncology, Pfizer, Pharmacyclics, Sigma Tau, Treadwell: Research Funding; AbbVie: Other: Travel reimbursement. OffLabel Disclosure: Ponatinib is approved for Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) that is resistant or intolerant to prior tyrosine kinase inhibitor therapy. In this study, we described outcomes with ponatinib in combination with hyperCVAD in the frontline setting, which is off-label.

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.

Background: As a single agent, rituximab produces an overall response rate (ORR) of 20-50% in patients with relapsed/refractory (r/r) B-cell non-Hodgkin lymphomas (NHL). Acquired resistance occurs, however, partially due to defective host immune effector mechanisms. Efforts to augment immune function have included combining rituximab with immune modulators such as IL-2, IL-12, interferonand lenalidomide. These efforts have resulted in improved efficacy of rituximab. Ipilimumab is a human IgG1κ monoclonal antibody (mAb) specific for the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152) expressed on activated T cells. CTLA-4 is a negative regulator of T-cell responses. Ipilimumab augments the immune response by disrupting the interaction of CTLA-4 with its ligands, B7-1 (CD80) and B7-2 (CD86). We therefore hypothesize that ipilimumab will augment the efficacy of rituximab. This phase I with an expansion cohort was designed to assess the safety of this combined therapy. Methods: Patients ≥ 18 years old with r/r CD20+ NHL, measurable disease, KPS ≥ 70%, neutrophils ≥ 1000, platelets ≥ 50,000 and adequate kidney and liver function were eligible. In this phase I with an expansion cohort, after establishing the recommended Phase II dose (RP2D), study patients would be randomized to differing treatment schedules of rituximab and ipilimumab. For induction in phase I, rituximab was given every week for four weeks and ipilimumab was administered every 3 weeks for 4 doses. Primary endpoints included toxicity and determination of the maximum tolerated dose (MTD)/RP2D during the first 7 weeks of induction of ipilimumab (2 doses) when given in combination with rituximab (4 doses, 375mg/m2 per dose). Secondary end points included immune response, ORR and progression free survival (PFS). In the phase I component, a standard 3+3 design was used to determine the MTD/RP2D. After the RP2D was established, 20 more patients would be randomized to one of two schedules: Arm A - ipilimumab on Day 1 together with the first dose of rituximab, or Arm B - ipilimumab on Day 15 together with the third rituximab dose. After induction in both the Phase I and the expansion cohorts, ipilimumab and rituximab were given every 12 weeks (+/- 1 week) on the same day for 1 year. Results: As of August 1 2015, 21 patients have been enrolled (1SLL, 9FL, 6DLCL, 1MCL, 4 indeterminate): 15 are evaluable for toxicity assessment at 7 weeks. The median age was 62 (32-71) and the median number of prior regimens was 2 (1-6). The RP2D of ipilimumab when given with rituximab (375 mg/m2) was 3 mg/kg, which was the highest dose tested. The most common grade 1-2 AEs (>20%) were:anemia (33%); diarrhea (20%); constipation (27%); nausea/vomiting (33%); fatigue (40%); anorexia (33%); low albumin (53%); hypocalcemia (33%); hyponatremia (27%). The most common grade 3-4 AEs (≥ 5%) were: anemia (20%); diarrhea (13%); leukopenia (13%); and hyponatremia (27%). There were no study related deaths. Twelve patients remain on treatment, 6 came off due to progression, 2 due to toxicity, and there was one death (unrelated). Conclusion: The RP2D of ipilimumab when given in combination with rituximab is 3 mg/kg. This dose is safe and moderately well tolerated. Enrollment continues in the expansion cohort to assess efficacy and the ability of ipilimumab to augment the rituximab-mediated immune response. Disclosures Off Label Use: Ipilimumab to augment rituxan in treating lymphoma. Chen:Merck: Consultancy, Research Funding; genentech: Consultancy, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding, Speakers Bureau.

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.

Turnip crinkle virions contain genomic RNA, Mr approximately 1.4 x 10(6), small RNA, Mr approximately 1.5 x 10(5) and a heterogeneous collection of intermediate size. Turnip crinkle virion RNA was translated in rabbit reticulocyte lysates into three major unrelated products of molecular weight 38,000 (p38, the virion coat protein), 36,000 (p36), and 25,000 (p25). The same three products, but in different proportion, were synthesized in wheat embryo extracts. Certain intermediate size RNA fractions translated into p38 and p36 products much more efficiently than did unfractionated RNA. The 1.5 x 10(5) molecular weight RNA did not induce amino acid incorporation. We propose a translation map of the TCV genome, based on tryptic analyses of the major products and of putative read-through proteins.

The amino acid sequence of BMV (brome mosaic virus) coat protein is presented and compared with the sequence derived from RNA structure. Apart from removal of the N-terminal methionine residue and acetylation of the following serine residue, no post-translational modifications occur.

Southern bean mosaic virus RNA was translated in wheat embryo extracts and in nuclease-treated rabbit reticulocyte lysates. Four principal products were synthesized: two related products with molecular weights of 105,000 and 75,000, a product with a molecular weight of 29,000 that closely resembled coat protein, and a product with a molecular weight of 14,000. Their proportion depended on ionic conditions and the translational system used.

A 3'-terminal oligonucleotide fragment, 161 bases long, can be obtained from each of the four brome mosaic virus RNAs by means of nuclease digestion. Like the four intact brome mosaic virus RNAs, each fragment accepts tyrosine in a reaction catalyzed by wheat germ aminoacyl-tRNA synthetase. The complete nucleotide sequence of the RNA 4 fragment has been determined by use of standard radiochemical methods. Comparative data for the fragments from RNAs 1, 2, and 3 show that they have nearly the same sequence as the RNA 4 fragment. The eight bases adjacent to the 3' terminus of the RNA 4 fragment are identical in sequence to the eight terminal bases of tyrosine tRNA from Torula utilis and eleven interior bases are identical in sequence to eleven bases encompassing the anticodon region of tyrosine tRNA from Saccharomyces cerevisiae, T. utilis, and Escherichia coli. Nevertheless, reasonable base-pairing schemes yield, at best, a distorted cloverleaf secondary structure.

AMV was purified by differential centrifugation and n -butanol chloroform-water extractions at 6°. Purified preparations exhibited three closely-spaced schlieren peaks in the analytical ultracentrifuge with sedimentation coefficients of 73, 89 and 99 S. The three fractions were partially separated from each other by the use of sucrose density-gradient techniques. Local-lesion assays demonstrated that infectivity was associated with the 99-S fraction. Precipitin endpoint, cross-absorption and gel-diffusion tests indicated that the three fractions were serologically similar. Electron micrographs showed that the particles were rod-like with diameters of 200 A and lengths 550 A or less. The 73-S fraction contained a greater proportion of particles shorter than 550 A than did the 99-S fraction. Approximately equal amounts of nucleic acid were associated with each of the three fractions. Attempts to show that the nucleic acid associated with the 73-S fraction was contributed by host organelles failed.

The RNA from bacteriophage Qβ can be translated by cell-free extracts from wheat embryos. This translation, by 80 S ribosomes, occurs at a low magnesium ion concentration. Three products are synthesized which coelectrophorese with Qβ proteins synthesized in Escherichia coli extracts. The smallest of these has been identified as coat protein. Although the polycistronic bacteriophage message is translated with fidelity, the efficiency is much less than when the monocistronic brome mosaic virus coat protein message is translated.

Unfractionated bromegrass mosaic virus RNA and each of its three naturally occurring components, of molecular weight 1.0 × 10 6 , 0.7 × 10 6 , and 0.3 × 10 6 , stimulate the incorporation of amino acids into peptides in an E. coli cell-free incorporating system. Analysis of tryptic digests by paper electrophoresis and chromatography indicated that the products induced by the RNA small component of molecular weight 0.3 × 10 6 , and by the unfractionated RNA were similar.

The RNA's from the bacteriophages M12 and R17, were digested with pancreatic ribonuclease to yield pyrimidine mononucleotides and pyrimidineterminated oligonucleotides. All the non-isomeric digestion products were separated by ion exchange chromatography. The complete set of such fragments has been designated the pyrimidine catalog of the RNA. M12 RNA and R17 RNA were found to have very similar pyrimidine catalogs and, by inference, very similar RNA sequences.

An electron microscopic examination has been made of thin sections of kidney mitochondria isolated in 0.44 M sucrose from vitamin D deficient rats and from vitamin D treated rats. Vitamin D deficiency resulted in swollen and morphologically damaged mitochondria characterized by large intercristal spaces and disrupted cristal systems. These changes were prevented by vitamin D.

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.

The protein and RNA of bromegrass mosaic virus were separated from each other by dialysis of the virus against 1 M -calcium chloride. In this medium the virus is degraded, the RNA is quantitatively precipitated and there remains a supernatant solution of viral protein. The protein obtained in this way was soluble in various buffers in the pH range 5·5 to 6·5 and exhibited a single symmetrical sedimenting boundary in the ultracentrifuge. Its molecular weight was 40,000 ± 2,000 as determined by the Ehrenberg method of approach to sedimentation equilibrium. Amino acid analyses indicated that there were about 52 lysyl plus arginyl residues per molecular weight of 40,000. Tryptic hydrolysis of urea-denatured protein yielded 23 soluble peptides and an insoluble residue amounting to about 20% by weight of the intact protein. From the number of soluble tryptic peptides and an assumed number of tryptic peptides in the insoluble residue it was concluded that the molecular weight of the protein subunit of bromegrass mosaic virus was slightly greater than 20,000 and that the protein moiety obtained by treatment with 1 M -CaCl 2 was a relatively stable dimer of the subunit. Partial separation of the soluble peptides resulting from tryptic hydrolysis was achieved on a preparative scale by column chromatography.

Broad bean mottle virus can be degraded by dialysis against 1 M -CaCl 2 . The virus RNA is quantitatively precipitated together with some of the protein. Most of the virus protein, free of RNA, can be recovered from the supernatant liquid. The amino acid composition of the protein was determined. End group analyses were performed on the oxidized protein. The C-terminal amino acid was found to be alanine. No N-terminal amino group was found. The tryptic peptides of the oxidized protein were separated by a two-dimensional combination of paper chromatography and electrophoresis. Twenty-eight peptides were detected. From the number of tryptic peptides and the amino acid composition, the molecular weight of the protein subunit was calculated to be 20,500.

Wild cucumber mosaic virus and two virus-like particles can be degraded markedly at high pH, e.g., pH 10 or at a low pH, e.g., pH 2.5. Virus protein subunits can be isolated from the degradation products by means of precipitation at pH 5.5. Ultracentrifuge analysis of the protein preparation thus obtained showed evidence of polydispersity under most of the solution conditions tested. These experiments indicated that the polydispersity was due to reactive groups which form hydrophobic interactions, hydrogen bonds, and disulfide bonds. The sedimentation rate of the principal component was approx. 2.0 S. Amino acid analysis was carried out on the protein from which the minimal molecular weight was calculated to be approx. 21500.

The RNA bacteriophage R17 when exposed to pH 10.5 is disrupted to yield a protein and products of alkaline hydrolysis of the viral RNA. The protein has an intrinsic sedimentation coefficient of 41.1 s and an intrinsic viscosity of 0.055 dl./g at 20 °C. It has a typical protein absorbancy spectrum and contains less than 1% RNA. These data, together with electron micrographs, indicate that the protein consists of a hollow protein shell similar in structure to the protein portion of R17.

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.

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 pH's. 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 ability of RNA from several plant viruses to bind a specific amino acid in a manner like tRNA is now well established. All members of the tymovirus group so far examined contain RNA which can bind valine1–3; all members of the bromovirus group bind tyrosine (ref. 4 and T.C.H., in preparation), and tobacco mosaic virus (TMV) RNA binds histidine5. The ubiquity of the reaction, and the similarity of the esterification to that of aminoacylation of tRNA indicates that a significant biological function for the property exists. A tRNA-like function such as that of a virus-specific tRNA, or a missense tRNA seems plausible. Although there is evidence that turnip yellow mosaic virus (TYMV) RNA, or a fragment containing the 3′ terminus of TYMV RNA, may donate valine to nascent polypeptides6, it has been established that tyrosine, bound to brome mosaic virus (BMV) RNA, is not donated to peptidyl material in a cell-free system from wheat embryo when directed by viral, plant, or synthetic messenger RNA (ref. 7).

In cell-free extracts derived from rabbit reticulocytes, encephalomyocarditis RNA can be translated completely, and the products can be processed extensively to give encephalomyocarditis virion proteins and several nonvirion proteins, including a genome-coded protein required for processing. The latter is probably a protease. Translation is very efficient. Under typical conditions, each EMC RNA is translated approximately eight times during a 3-h period. Kinetic analyses (time-course experiments, pulse-chase experiments, and pulse-stop experiments) have been used to determine the time of appearance of major products, and these times have been correlated with map positions. The gene for the putative protease is located near the middle of the genome downstream from the virion protein genes. Ribosomes can travel the length of encephalomycarditis RNA within 30 min, but there is a delay in their progress along the RNA at some point soon after they traverse the region coding for virion protein precursors. This delay results in the accumulation of precursors for a period of about 10 min before the putative protease is made and virion proteins (epsilon, alpha, and gamma) are released by proteolysis.

The structure and size of potato spindle tuber viroid (PSTV) were investigated by electron microscopy. PSTV appears to be composed of two single-stranded RNA components of different sizes, one linear and one circular. The linear component is about four times more common than the circular. Both components exist in collapsed, highly base-paired structures under nondenaturing conditions. Both components are sensitive to digestion by pancreatic ribonuclease but are resistant to snake venom phosphodiesterase. From length measurements of formylated and non-formylated PSTV RNA, the estimated molecular weight for the linear molecule is 110,000, and for the circular, it is 137,000.

Qβ replicase, in the presence of Mn ++ , can use various RNAs as templates for synthesis of full-length RNA products and can also use Qβ plus-strand RNA as template in the absence of host factors.

Encephalomyocarditis virus RNA induced a low level of amino acid incorporation in wheat embryo extracts, but the incorporation did not result in the formation of any viral proteins. However, if wheat embryo extracts were supplemented with a ribosomal wash fraction from rabbit reticulocytes, a large stimulation of amino acid incorporation and the formation of encephalomyocarditis virus-specific proteins were obtained. Among the proteins synthesized were capsid precursor proteins which can be processed by an encephalomyocarditis virus-coded protease to produce the viral capsid proteins.

Poliovirus RNA can be translated completely and accurately in rabbit reticulocyte lysates; the nascent poly-protein is processed to give primary products 1a, X, and 1b indistinguishable from those made in poliovirus-infected HeLa cells. The capsid precursor protein 1a is processed to form the capsid proteins VP0, VP1, and VP3, while the noncapsid precursor 1b is processed to form protein 2.

The four RNAs of brome mosaic virus induce substantial incorporation of amino acids into protein when used as messengers in a cell-free protein-synthesizing system derived from wheat embryo. RNA 4 is highly efficient as a monocistronic messenger for the viral coat protein. Acetate, derived from acetyl coenzyme A, is incorporated into the product made in vitro. Although RNA 3 also contains the coat-protein cistron, it induces synthesis mostly of a protein larger than coat protein. RNAs 1 and 2 also induce the synthesis of substantial amounts of protein other than coat protein. However, an equimolar mixture of RNAs 3 and 4 or of 1, 2, 3, and 4 induces synthesis of coat protein almost exclusively. This result suggests that the coat-protein cistron, when present as a monocistronic messenger, inhibits translation of all other viral messages.

The oligonucleotides resulting from ribonuclease digestion of 32P-labeled R17 RNA were separated on the basis of their chain length. The observed distribution was in agreement with statistical expectation. Three kinds of octanucleotides occurred. Knowing their specific radioactivity and the total radioactivity in all of the fractions, it was concluded that R17 RNA contains approximately 3342 nucleotide residues. The molecular weight of R17 RNA is thus approximately 1·1 × 106.

Ribonucleic acids from the bacteriophage R17 and from R17 amber mutant AmB2 have been digested with ribonuclease T1. Of the products isolated, only one was different. It codes for the first six amino acid residues of the viral coat protein. The probable base sequence of the wild-type oligonucleotide is CpUpUpCpUpApApCpUpUpUpApCpUpCpApGp.

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.

Plants infected with wild cucumber mosaic virus produce two virus-like particles (designated top component a and top component b) in addition to virus (designated bottom component). The bottom component and a mixture of top component a and top component b were isolated by means of sucrose-density gradient centrifugation. The molecular weights, determined by sedimentation and diffusion experiments, were 4.0·106 for top component a, 4.3·106 for top component b, and 7.0·106 for bottom component. These particles differ in their nucleic acid content. Top component a has no nucleic acid, top component b contains approx. 11% RNA, and bottom component contains approx. 35% RNA. The relative proportion of the 4 nucleotide bases in the RNA of top component b and in the RNA of bottom component are similar. Both have an unusually high proportion of cytosine. The amino acid compositions of the three components are quite similar.

Incubation of tyrosine with the RNA from brome-mosaic-virus under conditions favourable for aminoacylation of tRNA resulted in binding of tyrosine to the viral RNA. Sucrose-density-gradient centrifugation showed that tyrosine binds to three and probably to all four of the viral RNA components. The bound amino acid was readily released in mild alkaline solutions. Of 20 amino acids tested, only tyrosine was found to bind. The maximum extent of binding observed was 0.58mol of tyrosine/mol of brome-mosaic-virus RNA.

Physical studies were carried out on RNA isolated from turnip yellow mosaic virus by extraction with phenol in the presence of sodium dodecyl sulfate and bentonite. Its molecular weight from sedimentation and viscosity measurements is about 1·9 × 10 6 , corresponding to one RNA molecule per virus particle. The RNA, immediately after isolation, was relatively compact, and unfolded to a less compact but stable form upon heating or alcohol-precipitation and subsequent dialysis. The RNA molecules exhibited a smaller hydrodynamic volume upon lowering the temperature without any significant change in the extinction coefficient. Under acidic conditions, the RNA molecules become more compact and less susceptible to temperature-induced unfolding; this has been ascribed to the formation of poly C-like structures. Polyamines such as bis(3-aminopropyl)-amine and spermine effected a marked reduction in the hydrodynamic volume of the RNA and increased its resistance to temperature-induced unfolding. Denaturation treatments were carried out on the RNA to detect hidden breaks and the possible presence of subunits. It was concluded that the RNA does not have subunits, although occasionally discrete degradation components were obtained.

Bacteriophage Qβ can be separated easily from several other RNA-containing bacteriophages by electrophoresis on large-pore acrylamide gels. The mobilities of these viruses depend both upon the acrylamide and cross-linker concentrations. Qβ has a complex electrophoretic pattern due to aggregation. An electrophoretic mutant of Qβ (called QβE1) has been isolated and partially characterized. The proteins of Qβ, QβE1, R17, and G17 have been characterized by acrylamide gel electrophoresis. Two continuous SDS-containing systems have been used: an SDS-urea-phosphate gel at pH 7.6 and an SDS-urea-phosphate gel at pH 11.9. On these, the coat proteins of Qβ and R17 and also their A proteins are separable because they differ in molecular weight. The molecular weight of the A protein of Qβ is 37,600 daltons. The coat protein of Qβ and its electrophoretic mutant QβE1 are also distinguishable though they differ by only a single charged amino acid. A low pH buffer system containing formic acid and urea but lacking SDS separates the coat proteins of Qβ, QβE1, and R17 readily.

Purified preparations of squash mosaic virus contain three kinds of macromolecular particles with sedimentation rates 57 S, 95 S and 118 S. The 118 S particle is infectious. The particles were separated from each other by means of density-gradient sedimentation. Their diffusion coefficients and amino acid composition were found to be similar. The particles differ mainly in their content of ribonucleic acid. The 57-S particle contains no RNA, the 95-S particle contains 27% RNA, the 118-S particle contains 35% RNA. The base compositions of the latter two RNA's are similar. Partial specific volumes were determined for the three particles and are consistent with values calculated from their RNA content and amino acid composition. The particles have molecular weights 4.5·106, 6.1·106 and 6.9·106, respectively.

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.

The bacteriophages R17 and M12 were grown in quantity and purified. They responded similarly to growth and purification conditions. Both appeared homogeneous in the ultracentrifuge. They were found to be serologically similar. The protein moieties of R17 and M12 were isolated by extraction with 67% acetic acid. R17 protein had greater electrophoretic mobility than M12 protein. R17 contained one more mole of lysine residues and one less mole of glutamic acid or glutamine residues than M12 per 14·2 × 10 3 g protein. Otherwise, their amino acid compositions were indistinguishable. Analysis of the alkali spectra of the proteins showed that each contained two moles of tryptophan residues per 14·2 × 10 3 g protein. R17 and M12 proteins were digested with trypsin and the products isolated, using a combination of paper chromatography and paper electrophoresis. R17 was found to have one more lysyl peptide than M12. Of the fourteen R17 peptides isolated, 11 were found to be identical in amino acid composition and two very similar to the corresponding 13 M12 peptides isolated. Comparison of the number of lysine and arginine residues per unit weight of the proteins with the number of major lysyl and arginyl tryptic peptides indicated that the chemical subunit weight of R17 and Ml2 coat proteins is about 14·2 × 10 3 .