Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells.

Cancer-associated fibroblasts (CAFs) comprise the majority of the tumor bulk of pancreatic ductal adenocarcinomas (PDACs). Current efforts to eradicate these tumors focus predominantly on targeting the proliferation of rapidly growing cancer epithelial cells. We know that this is largely ineffective with resistance arising in most tumors following exposure to chemotherapy. Despite the long-standing recognition of the prominence of CAFs in PDAC, the effect of chemotherapy on CAFs and how they may contribute to drug resistance in neighboring cancer cells is not well characterized. Here, we show that CAFs exposed to chemotherapy have an active role in regulating the survival and proliferation of cancer cells. We found that CAFs are intrinsically resistant to gemcitabine, the chemotherapeutic standard of care for PDAC. Further, CAFs exposed to gemcitabine significantly increase the release of extracellular vesicles called exosomes. These exosomes increased chemoresistance-inducing factor, Snail, in recipient epithelial cells and promote proliferation and drug resistance. Finally, treatment of gemcitabine-exposed CAFs with an inhibitor of exosome release, GW4869, significantly reduces survival in co-cultured epithelial cells, signifying an important role of CAF exosomes in chemotherapeutic drug resistance. Collectively, these findings show the potential for exosome inhibitors as treatment options alongside chemotherapy for overcoming PDAC chemoresistance.

Cell-to-cell movement of beet necrotic yellow vein virus: I. Heterologous complementation experiments provide evidence for specific interactions among the triple gene block proteins.

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.

Location of the cistron of the tobacco mosaic virus coat protein.

Treatment of tobacco mosaic virus (TMV) RNA with T1 RNase under mild conditions cuts the RNA molecule into a large number of fragments, only a few of which may be specifically recognized by disks of TMV protein. It has been shown elsewhere that these specifically recognized RNA fragments are a part of the coat protein cistron, the portion coding for amino acids 95 to 129 of the coat protein. It is reported that different size classes of partially uncoated virus particles were prepared by limited reconstitution between TMV RNA and protein or by partial stripping of intact virus with DMSO. Both procedures produce nucleoprotein rods in which the 5'-terminal portion of the RNA is encapsidated and the 3'-terminal region is free. The free and the encapsidated portions of the RNA were each tested for the ability to give rise to the aforesaid specifically recognized fragments of the coat protein cistron upon partial T1 RNase digestion. It was found that only the 3'-terminal third of the virus particle need to be uncoated in order to expose the portion of the RNA molecule from which these fragments are derived. We conclude, therefore, that the coat protein cistron is situated upon the 3'-terminal third of the RNA chain, i.e. within 2000 nucleotides of the 3'-end.

Assembly of tobacco mosaic virus in vitro: effect of state of polymerization of the protein component.

The in vitro assembly of tobacco mosaic virus from its constituent RNA and protein was followed by methods of electron microscopy. The effect of the state of polymerization of the protein upon the initiation of assembly of tobacco mosaic virus rods, and the subsequent rod elongation, was investigated. Protein in two identifiable states of polymerization was used: the 20S "disc", consisting of 34 monomers arrayed as a two-ring structure, and the 4S "A-protein", consisting of polymers in the trimer range of size. It is concluded, in confirmation of results of others, that rod assembly is initiated by the attachment of one end of the RNA chain of tobacco mosaic virus to one (or possibly a few) disc structure. Rod elongation, on the other hand, is found to take place by the sequential addition of structures of the size of A-protein, or smaller, to the previously initiated rods.

Observations concerning the discontinuous DNAs of cauliflower mosaic virus.

The double-stranded circular DNA encapsidated within cauliflower mosaic virus (CaMV) particles contains three single-stranded discontinuities, two in one strand and one in the other, so that, upon denaturation, three linear single-stranded DNAs are produced. Here we show that a fourth much smaller single-stranded DNA, termed alpha1, is also present in denatured CaMV DNA preparations. The 5' extremity of alpha1 is identical to that of the alpha-strand, the strand of DNA possessing only one interruption, while its 3' extremity lies just two nucleotides downstream from a major transcription initiation site. We also show that the interrupted strand at each discontinuity sometimes has a single ribonucleotide in place of a deoxyribonucleotide at its 5' extremity. Oligoribonucleotide chains of eight and 10 residues in length have also been detected at the 5' end of one of the discontinuities. These structures are thought to be the vestiges of primers which have not been completely excised prior to encapsidation of the DNA. The possibility that synthesis of the alpha-strand occurs by reverse transcription of viral RNA using initiator tRNA(met) as primer is discussed.

Specific encapsidation of fragments of TMV RNA.

The in vitro reconstitution of tobacco mosaic virus (TMV) is initiated by the binding of a disk of TMV protein to the 'disk recognition site', a region of the RNA chain at or near the 5'-terminus for which the disk has special affinity. In order to gain insight into the recognition process, we have studied the ability of disks to encapsidate short RNA fragments produced by partial pancreatic or T1 RNase digestion of TMV RNA. The disk is capable of dicriminating among such fragments, encapsidating only a few of the many present in the digest. The products of encapsidation are short nucleoprotein rods of the same diameter as TMV and of length proportional to that of the encapsidated RNA fragment. The particles differ from TMV, however, in one significant aspect (apart from their length): they possess rings of RNA-free protein at one or both extremities of the rod. In the case of T1 RNase digestion the principal encapsidated fragments were fragments T1 (105 nucleotides) and a family of smaller fragments containing elements of the same sequence. Partial digestion with pancreatic RNase generated only one major fragment (fragment P1; 150 nucleotides) with affinity for the disk. Fragment T1 has been sequenced and shown to represent a portion of the coat protein cistron. Fragment P1 has been partially sequenced but its function is not yet known. Several lines of evidence indicate that fragment T1 is not the disk recognition site. The portion of the TMV RNA chain from which fragment P1 is derived, on the other hand, is encapsidated early in the reconstitution process; thus fragment P1 may contain the disk recognition site. Fragment T1 and fragment P1 both have purine-rich and cytosine-poor sequences near their termini. In addition, fragment T1, and possibly fragment P1, possess a periodicity of order three in purine residues. It seems likely that one or both of the aforesaid properties are largely responsible for the affinity of these fragments for the disk.

Sequence of a specifically encapsidated RNA fragment originating from the tobacco-mosaic-virus coat-protein cistron.

When 25-S tobacco mosaic virus (TMV) protein aggregate and TMV RNA, which has been partially digested by T1 RNase, are mixed under conditions suitable for reconstitution, only a few RNA fragments are encapsidated. These fragments were isolated and purified by polyacrylamide gel electrophoresis. The sequence of the three main fragments, the longest of which (fragment 1) was estimated to contain 103 nucleotides, has been determined. The two smaller fragments are portions of the longer chain produced by an additional specific scission. Because of the great affinity of 25-S TMV protein for this nucleotide sequence, it will be referred to as the "specifically encapsidated RNA fragment". The occurrence of a "hidden break" in the sequence has been demonstrated: fragment 1, purified by electrophoresis on a polyacrylamide gel without 8 M urea, gives rise upon further electroporesis in the presence of urea to two new bands corresponding to the two halves of the molecule. A stable hair-pin secondary structure has been derived from the base sequence which can account for the specificity of action of the enzyme. Because of its properties, we have suggested elsewhere that the sequence of fragment 1 might correspond to the disk recognition site for reconstitution, which is known to be located at the 5' end of the intact RNA. But experiments with TMV RNA whose 5'-OH end has been radioactively phosphorylated with polynucleotide kinase show that this is not the case. Analysis of the amino acid coding capacity of the fragment has instead revealed that fragment 1 is a portion of the TMV coat protein cistron.

Observations concerning the sequence of two additional specifically encapsidated RNA fragments originating from the tobacco-mosaic-virus coat-protein cistron.

The incubation of 25-S tobacco mosaic virus (TMV) protein with a mixture of RNA fragments produced by partial T1 RNase digestion of TMV RNA results in the encapsidation of only a few species of RNA. In addition to the most predominant species, fragment 1, whose sequence has been described in the prededing paper, two other species, fragment 41 and fragment 21 are coated by the protein. These two RNA fragments were purified by polyacrylamide gel electrophoresis and subjected to total digestion with pancreatic and T1 RNase. The oligonucleotides were separated by paper electrophoresis and characterized insofar as possible by digestion with the complementary ribonuclease. From the amino acid coding capacity of the oligonucleotides liberated from fragments 41 and 21 by T1 RNase digestion, it appears that these two fragments, like fragment 1, are derived from the coat protein cistron. They are situated immediately prior to fragment 1 and, together with this fragment, consitute a continuous stretch of 232 nucleotides of the cistron which codes for animo acids 53 to 130 of the coat protein. The order of the fragments in the sequence is 21-41-1. A possible model for the secondary structure of this portion of the sequence is proposed.

Nucleotide sequence of beet mild yellowing virus RNA.

The complete nucleotide sequence of the genomic RNA of beet mild yellowing virus, isolate 2ITB, is reported. The RNA consists of 5722 nucleotides and contains six long open reading frames which conform to the arrangement characteristic of Subgroup 2 luteoviruses. The three 3'-proximal open reading frames, which encode the viral coat protein, a putative movement protein and the Readthrough Domain, are highly homologous to the corresponding genes of beet western yellows luteovirus while the three 5'-proximal open reading frames are more closely related to the corresponding genes of cucurbit aphid borne yellows luteovirus. The sequence data thus indicate that beet mild yellowing virus should be considered a distinct virus rather than a strain of beet western yellows virus.

Sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA.

The sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA has been determined. The sequence contains the entire coat protein cistron as well as regions to its left and right. Sequence characterization was by conventional methods for use with uniformly 32P labeled RNA complemented by newer methods for in vitro 5' and 3' 32P end-labeling of RNA and its subsequent rapid analysis. The noncoding region separating the coat protein cistron from the 3' terminus is 204 residues long and may be folded into a clover-leaf-type secondary structure. The distribution of termination codons to the left of the coat protein cistron suggests that the end of the adjacent cistron is separated from the beginning of the coat protein cistron by only two nucleotides. The subgenomic viral coat protein mRNA was isolated from infected tissue and shown to be capped. The nontranslated sequence separating the cap from the AUG initiation codon is 9 residues long and thus overlaps a portion of the adjacent cistron on the genome RNA.