Virus Subgenomic RNA Research Articles

Potato Leaf Roll Virus 3' Genome Organization: Sequence of the Coat Protein Gene and Identification of a Viral Subgenomic RNA

(...) The coat protein gene contains 624 nucleotides and encodes a protein of 208 amino acids with a calculated molecular weight of 23.2 kDa. This gene is located internal to the 3'-end of the genome, contains the complete overlap of an open reading frame (ORF) encoding a 17.4 kDa protein, and is followed in-frame by an ORF encoding a 56.4 kDa protein. Northern blot hybridization experiments have identified a 3'-positioned viral subgenomic RNA produced in PLRV-infected plants. This subgenomic RNA is not encapsidated and, by analogy to other RNA plant viruses, is likely the mRNA for expression of the 3'-portion of the genome (...)

A tobacco mosaic virus-hybrid expresses and loses an added gene

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3′ region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.

A measles virus subgenomic RNA: Structure and generation mechanism

A plaque-derived line of measles virus induced the synthesis of 21S subgenomic RNA in addition to 50S genomic RNA. The nucleotide sequence of the former RNA was determined in comparison with that of the latter. The 21S subgenomic RNA was 2646 nucleotides long and had a copyback structure containing a 50S genomic 5′-end. At the site where copying turns back, there was a 15-nucleotide-long sequence resembling the “measles virus consensus sequence” which is present at the beginning of each gene on the measles virus genome and is presumed to serve as a signal for the initiation of transcription. Recognition of the specific sequence may have been involved in the aberrant replication and may represent an additional and novel copy choice mechanism leading to the generation of subgenomic RNAs.

Identification of the 5′ end of the rubella virus subgenomic RNA

The 5′ end of the subgenomic RNA of rubella virus was determined by primer extension. The Maxam-Gilbert sequence ladder of the primer extension product contained a determinable sequence which was colinear with the complement of the sequence of the genomic RNA through nucleotide 3325 from the 3′ end of the genomic RNA and a pair of bands in every lane above the determinable sequence. These results indicated that synthesis of the subgenomic RNA is initiated internally on the minus-polarity genome-length RNA template and that the length of subgenomic RNA is 3327 nucleotides excluding the poly(A) tail. There are thus 77 nucleotides between the 5′ end of the subgenomic RNA and the first AUG, which is the initiation codon for the structural protein open reading frame. The initiation site of rubella virus subgenomic RNA synthesis is 20 nucleotides downstream from a block of 28 nucleotides which shares homology with the nucleotide sequence which is conserved at the subgenomic RNA initiation site in the , the other genus of Togaviruses.

A centrifugation method for separation of plant viral genomic and subgenomic RNAs

Using alfalfa mosaic virus (AMV) as a model, a simple method for separating plant viral genomic RNAs from their subgenomic counterparts was established. The method relies on sucrose gradient fractionation under carefully selected conditions of centrifugation and fraction collection. The RNA components are recovered in nearly quantitative yield and have full biological activity as measured by infectivity of the reconstituted RNAs in suitable protoplasts and plant hosts. The individual RNAs, on the other hand, show no such infectivity, indicating that the separation is indeed complete.

Generation of defective-interfering particles by rubella virus in vero cells

The generation of defective-interfering (DI) particles by rubella virus during serial undiluted passage and persistent infection in Vero cells was studied. A series of 24 serial undiluted passages was initiated with plaque-purified virus. The virus titer remained relatively constant through the first nine passages, after which it declined, reaching a low level of 20-fold less than the originating stock by passage 15. In subsequent passages, the titer cycled. Intracellular DI RNAs were first detectable at passage 4, at which time DI RNAs of 7500 and 1400 nucleotides in length were observable. Thus, the rate of which DI RNAs were generated by rubella virus during serial undiluted passage was similar to the rate of DI generation by other enveloped RNA viruses during serial undiluted passage. The longer rubella DI RNA was present in all passages subsequent to passage 4, while the 1400-nucleotide DI RNA was replaced by a DI RNA of 800 nucleotides in length by passage 15. Subsequent to passage 7, the relative amount of genomic RNA declined dramatically and the DI RNAs became the predominant intracellular virus-specific RNA species. Negative-polarity RNA species corresponding to the 7500- and 800-nucleotide DI RNA species were indentified. The 7500- and 1400-nucleotide DI RNA species were encapsidated into virus particles while the presence of the 800-nucleotide DI RNA species in virus particles could not be detected. Interestingly, the rubella virus subgenomic RNA was present in virus particles in preparations containing DI RNAs. A persistent infection was initiated by subculturing the surviving cells from a high multiplicity of infection with plaque-purified virus. Intracellular DI RNAs were first detectable at Day 19 after initiation of persistence and became significant by Day 26. The amount of genomic RNA began to decrease at Day 47 and was undetectable after Day 68. Through Day 54, there were several DI RNA species present, but at later times, one of these species became predominant. Thus, DI particles were generated during persistent infection, but their presence was not necessary for initiation of persistence.

Temporal regulation of bovine coronavirus RNA synthesis

The structure and synthesis of bovine coronavirus (BCV)-specific intracellular RNA were studied. A genome-size RNA and seven subgenomic RNAs with molecular weights of approximately 3.3 × 10 6, 3.1 × 10 6, 2.6 × 10 6, 1.1 × 10 6, 1.0 × 10 6, 0.8 × 10 6 and 0.6 × 10 6 were detected. Comparisons of BCV intracellular RNAs with those of mouse hepatitis virus (MHV) demonstrated the presence of an additional RNA for BCV, species 2a, of 3.1 × 10 6 daltons. BCV RNAs contain a nested-set structure similar to that of other coronaviruses. This nested-set structure would suggest that the new RNA has a capacity to encode a protein of approximately 430 amino acids. Kinetic studies demonstrated that the synthesis of subgenomic mRNAs and genomic RNA are differentially regulated. At 4 to 8 h post-infection (p.i.), subgenomic RNAs are synthesized at a maximal rate and represent greater than 90% of the total viral RNA synthesized, whereas genome-size RNA accounts for only 7%. Later in infection, at 70 to 72 h p.i., genome-size RNA is much more abundant and accounts for 88% of total RNA synthesized. Immunoprecipitations of [ 35S]methionine-pulse-labeled viral proteins demonstrated that viral protein synthesis occurs early in the infection, concurrent with the peak of viral subgenomic RNA synthesis. Western blot analysis suggests that these proteins are stable since the proteins are present at high level as late as 70 to 72 h p.i. The kinetics of production of virus particles coincides with the synthesis of genomic RNA. These studies thus indicate that there is a differential temporal regulation of the synthesis of genomic RNA and subgenomic mRNAs, and that the synthesis of genomic RNA is the rate-limiting step regulating the production of virus particles.

Time course of virus-specific macromolecular synthesis during rubella virus infection in vero cells

Virus specific macromolecular synthesis was studied in Vero cells infected with plaque-purified rubella virus under one-step multiplication conditions. Under these conditions, the rate of virus production was found to increase rapidly until 24 hr postinfection after which time the rate of virus production rose more slowly, reaching a peak level at 48 hr postinfection. This peak rate of virus production was maintained through 72 hr postinfection. A majority of the cells remained alive through 96 hr postinfection, although a 20 to 30% decrease in the number of living cells occurred between 24 and 48 hr postinfection, the time period at which cytopathic effect was first observed. The virus structural proteins were first detected intracellularly at 16 hr postinfection. The rate of synthesis of these proteins was already maximal at 16 hr postinfection and remained constant through 48 hr postinfection. By immunofluorescence, cells expressing virus proteins were first observed at 12 hr postinfection. At 24 hr postinfection, 35 to 50% of the cells in the infected culture were exhibiting immunofluorescence, at 36 hr postinfection, 65 to 90% of the cells were exhibiting immunofluorescence, and at 48 hr postinfection, all of the cells were exhibiting immunofluorescence. The virus genomic and subgenomic RNA species were first detectable by 12 hr postinfection. The rate of synthesis of both of these species peaked at 26 hr postinfection. Rubella virus infection was found to have no effect on total cell RNA synthesis. However, a modest inhibition of total cell protein synthesis which reached 40% by 48 hr postinfection was observed. When Northern analysis of RNA extracted from infected cells was performed, a negative-polarity, virus-specific RNA probe hybridized only to the virus genomic and subgenomic RNA species. A positive-polarity, virus-specific RNA probe hybridized predominantly to a negative-polarity RNA of genome length indicating that both the genomic and subgenomic RNAs are synthesized from a genome-length negative-polarity template. Defective interfering (DI) RNAs were not detected in infected cells through 96 hr postinfection or in cells onto which virus released through 96 hr postinfection was passaged. Thus, the generation of DI particles by rubella virus appears to play no role in the slow, noncytopathic replication of this virus or in the ability of rubella virus-infected cells to survive for extended periods of time.

N-methylisatin-beta-4',4'-diethylthiosemicarbazone, an inhibitor of Moloney leukemia virus protein production: characterization and in vitro translation of viral mRNA.

The mode of inhibition of N-methylisatin-beta-4',4'-diethylthiosemicarbazone (M-IBDET) on Moloney leukemia virus production was studied. Drug treatment of infected cells did not alter the amounts or sizes of the 35S and 22S subgenomic viral RNAs. The translation abilities of poly(A)+ RNA derived from M-IBDET-treated cells was also unaffected, as judged by cell-free translation analysis. Poly(A)+ RNA derived from M-IBDET-treated cells directed translation of equal amounts of viral gag precursors, gPr-80gag and Pr-65gag, as did poly(A)+ RNA prepared from untreated cells. The addition of M-IBDET to a cell-free translation system programmed with either total poly(A)+ RNA extracted from infected cells or hybrid-selected viral RNA inhibited the synthesis of viral protein precursors. An examination of the effect of M-IBDET on polysomes engaged in the translation of viral proteins revealed a fourfold accumulation of polysomal virus-specific RNA in drug-treated cells. These results suggest that the inhibition of Moloney leukemia virus by M-IBDET involves a block in the translation of viral RNA rather than interference with viral RNA transcription.

Replicative events in hepatitis A virus-infected MRC-5 cells

Replication of hepatitis A virus (HAV) in MRC-5 cells was studied under one-step growth conditions. Viral replication neither interfered detestably with cellular DNA, RNA, and protein synthesis, nor could cytopathologic changes be recorded over a prolonged period of incubation. Synthesis of mature, infectious HAV particles could be detected as early as 2–4 days p.i. and occurred at a maximal rate around 8 days p.i., shortly before infectivity titers reached a plateau. Synthesis of total viral RNA, of positive-strand genomic RNA, of viral mRNA, as well as of negative-strand RNA followed the same pattern. By Day 14 p.i., when active HAV replication had developed into persistent infection, synthesis of viral RNA declined to background levels. The mechanism(s) guiding active HAV replication into a state of persistent infection could not be positively defined. Yet there exists the possibility that this is brought about by down regulation of viral RNA synthesis. Whether this is related to the appearance of a subgenomic viral RNA molecule about 2000 nucleotides in length and detected in association with ribosomes on Days 7 and 10 p.i. remains to be shown.

High resolution mapping of carnation mottle virus-associated RNAs

The genomic coordinates from which carnation mottle virus (CarMV) subgenomic RNA 5′-ends originate have been determined by high resolution S, nuclease protection and primer-extension mapping techniques. The two, 3′-proximal subgenomic RNAs initiate at nucleotides 2532 and 2315 (counting from the genome 5′ terminus). These RNAs contain 1472 and 1689 nucleotides, respectively. There is substantial nucleotide sequence homology surrounding the transcriptional start sites of the two subgenomic RNAs. The 1.5-kb RNA uses the first AUG to initiate translation of viral coat protein. The first AUG on the 1.7-kb RNA begins a short open reading frame of 183 nucleotides. This region encodes a M r = 6746 polypeptide found by in vitro translation of authentic subgenomic RNA as well as synthetic SP6 transcripts. By SP6 transcription and cell-free translation, we have also mapped coding sequences for a previously identified CarMV gene product, p27, to the 5′ terminus of the viral genome.

Characterization of a myc-containing retrovirus generated by propagation of an MH2 viral subgenomic RNA.

We have previously isolated, from wild-type MH2 virus that contains the two oncogenes mil and myc, mutants defective in one or the other oncogene product. We report here the molecular cloning and extensive characterization of MH2 CL25 provirus lacking the v-mil oncogene. Our results indicate that this virus corresponds to the propagation of the 2.8-kilobase subgenomic RNA of MH21.

In Vitro viral RNA synthesis by a subcellular fraction of TMV-inoculated tobacco protoplasts

A subcellular fraction which can synthesize viral RNA and subgenomic RNA in vitro was prepared from tobacco mosaic virus (TMV)-inoculated tobacco protoplasts. S 1-Resistant fragment analysis with strand specific TMV cDNA showed that a large amount of plus-stranded and a small amount of minus-stranded, genome-size RNA was synthesized by this subcellular fraction. Plus-stranded subgenomic RNA of coat protein mRNA size was also synthesized. The time course of the appearance of viral RNA synthetic activity was consistent with that of the appearance of TMV infectivity in vivo.

Novel polypeptides encoded by influenza virus subgenomic (DI type) virion RNAs

We have isolated a ts mutant of influenza A/FPV/Rostock/34 that induces the synthesis of a novel small polypeptide in infected cells. This polypeptide is encoded by a subgenomic virion RNA derived from RNA segment 3, apparently by internal deletion. A second polypeptide, similarly derived from RNA segment 1, was found only after in vitro translation of infected cell RNA. The subgenomic vRNAs we describe are probably similar to those found in influenza DI virus preparations. The possible role of ‘subgenomic’ polypeptides in DI virus-mediated interference is discussed.

Characterization of the cell-free translation products of carnation mottle virus genomic and subgenomic RNAs

The in vitro translation products of carnation mottle virus (CarMV) genomic and subgenomic RNAs were analysed using a rabbit reticulocyte lysate system. Viral RNAs directed synthesis of three main polypeptides, p80, p40, and p34. p40, which was the predominant product using unfractionated virion RNA as template, was identified as coat protein based on electrophoretic mobility through SDS-polyacrylamide gels and immunoprecipitation with anti-CarMV serum. Upon size-fractionation of virion RNAs by sucrose gradient centrifugation and subsequent translational analysis, p40 was found to be encoded by subgenomic RNA, whereas p80 and p34 were synthesized from templates of genome length. p80 and p34 were found to contain common or overlapping amino acid sequences by analysis of cleavage peptides formed during proteolysis with α-chymotrypsin. These data support CarMV translational mechanisms other than those proposed in a previous study (R. Saloman, M. Bar-Joseph, H. Soreq, I. Gozes, and U. Z. Littauer, 1978, Virology 90, 288–298).

Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus.

The 9,213-nucleotide structure of the AIDS/lymphadenopathy virus has been determined from molecular clones representing the integrated provirus and viral RNA. The sequence reveals that the virus is highly polymorphic and lacks significant nucleotide homology with type C retroviruses characterized previously. Together with an analysis of the two major viral subgenomic RNAs, these studies establish the coding frames for the gag, pol and env genes and predict the expression of a novel gene at the 3' end of the genome unrelated to the X genes of HTLV-1 and -II.

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA.

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.

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.

Plant Virus-Specific Transport Function And Resistance Of Plants To Viruses

Publisher Summary The transport of infection is a distinct virus-specific function—transport function (TF)—performed by virus-coded (or virus-induced) proteins. The TF can be complemented in mixed infection with a helper virus; a temperature-resistant helper assists the systemic spreading of the virus temperature-sensitive in TF. Complementation can be achieved with some unrelated viruses—that is, the TF is nonspecific. Systemic spreading of infection takes place by two ways: (1) slow cell-to-cell movement (short distance transport) in the parenchyma and (2) rapid migration over long distances via the conducting tissues (long-distance transport). The long-distance transport of plant viruses occurs usually in the phloem and, in a few cases, in the xylem. The rate of virus transport in the infected tissue appears to be determined by the number of plasmodesmata connecting the neighboring cells; the increase in the number or size or structural alterations of plasmodesmata may favor enhanced transport of infection. Resistance of nonhost plants to viruses can be because of the impossibility of the infection transport (subliminal infection) and the virus being confined to the primarily infected cells. The virus genetic material is transported through the infected plant in the form of special transport particles—virus-specific informosome-like ribonucleoproteins (vRNP) differing from mature virions. These vRNP contain viral genomic and subgenomic RNAs and virus-specific and cell proteins.

Black Beetle Virus: Messenger for Protein B Is a Subgenomic Viral RNA

Black beetle virus induces the synthesis of three new proteins, protein A (molecular weight, 104,000), protein alpha (molecular weight, 47,000), and protein B (molecular weight, 10,000), in infected Drosophila cells. Two of these proteins, A and alpha, are known to be encoded by black beetle virus RNAs 1 and 2, respectively, extracted from virions. We found that RNA extracted from infected cells directed the synthesis of all three proteins when it was added to a cell-free protein-synthesizing system. When polysomal RNA was fractionated on a sucrose density gradient, the messengers for proteins A and alpha cosedimented with viral RNAs 1 (22S) and 2 (15S), respectively. However, the messenger for protein B was a 9S RNA (RNA 3) not found in purified virions. Like the synthesis of viral RNAs 1 and 2, intracellular synthesis of RNA 3 was not affected by the drug actinomycin D at concentrations which blocked synthesis of host cell RNA. This indicated that RNA 3 is a virus-specific subgenomic RNA and, therefore, that protein B is a virus-encoded protein.