Nonrandom packaging of host RNAs in moloney murine leukemia virus

Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA(Met) were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Psi- mRNAs and some other host RNAs may be specifically excluded from assembly sites.     

Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes

A single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal ("Psi") in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and Psi- particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed.     

tRNA''s associated with the 70S RNA of avian myeloblastosis virus.

The distribtuion of various amino acid tRNA's in the 4S RNA components of avian myeloblastosis virus (AMV) and in 4S RNA prepared from chicken cmbryo cells, chicken myeloblasts, and chicken livers was determined. This was done by aminoacylating the 4S RNA samples with a mixture of 17 radioactive amino acids and subsequently identifying the tRNA-accepted amino acids on an amino acid analyzer after deacylation. In embryo cells, myeloblasts, and liver, tRNA's accepting all 1m amino acids were demonstrated. "Free" AMV 4S RNA was characterized by very low quantities of glutamate, valine, and tyrosine tRNA's. RNAs accepting all 17 amino acids, with the exception of tyrosine, were shown to be present in the "70S-associated" 4S RNA which dissociates at 60 C. The bulk of the 70S-associated 4S RNA was dissociated at 60 C at low ionic strength with a concomitant conversion of 70S RNA to 35S RNA. The residual associated 4S RNA was dissociated by further heating of the 35S RNA to 80 C; tryptophan tRNA accounted for greater than 90% of the total amino acid accepting activity in this fraction. The results support other studies in suggesting that tryptophan tRNA may serve as a primer for DNA synthesis in AMV, as has been shown in Rous sarcoma virus.     

Subgenomic mRNA of Aura alphavirus is packaged into virions.

Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.     

Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1)

Gag proteins of human immunodeficiency virus type 1 (HIV-1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA-binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV-1 gag proteins, the packaging signal, psi and nucleolin affect the budding of HIV-1. Here we report that nucleolin enhances the release of HIV-1 virions which contain psi. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV-1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV-1.     

Interferon treatment of Ehrlich ascites tumor cells: effects on exogenous mRNA translation and tRNA inactivation in the cell extract.

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S60INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30INT between tRNA from interferon-treated cells and tRNA from control cells. Futhermore, no difference was found in the rate of inactivation in S30INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under out conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation fo exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Transcription of DNA from the 70S RNA of Rous Sarcoma Virus II. Structure of a 4S RNA Primer

The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus. We purified these primers in three different ways-by isolation of the covalent complex between primer and nascent DNA, by differential melting of the 70S RNA, and by two-dimensional electrophoresis in polyacrylamide gels. The 4S RNAs purified by these procedures were homogeneous and possessed very similar if not identical nucleotide compositions and sequences. The RNAs were approximately 75 nucleotides long, had pG at the 5' terminus and CpCpA(OH) at the 3' terminus, and contained a number of minor nucleotides characteristic of tRNA. In contrast to most tRNA's, the primer lacked rTp and contained Gp (Psip, Psip, Cp) Gp (possibly in place of the characteristic sequence GprTpPsipCpGp). At least 50% of the 4S primers available on 70S RNA were utilized in a standard polymerase reaction in vitro.     

Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S30_INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30_INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30_INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30_INT between tRNA from interferon-treated cells and tRNA from control cells. Furthermore, no difference was found in the rate of inactivation in S30_INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under our conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation of exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

RNA-DNA Covalent Bonds Between the RNA Primers and the DNA Products Formed by RNA Tumor Virus DNA Polymerase

Initiation of DNA synthesis by endogenous RNA primer molecules was studied with three different RNA tumor viruses. The influence of the method of virus disruption on the observed RNA-DNA bonds was ascertained. Ether disrupted virions of both murine leukemia virus (MuLV) and the B77 strain of avian sarcoma virus (B77 virus) have rC-dC and rA-dA covalent linkages between RNA primers and newly synthesized DNA. None of the 14 other possible bonds were formed. Ether-disrupted virions of avian myeloblastosis virus (AMV) have rU-dC and rA-dA linkages. In contrast, work reported herein and from other laboratories shows that Nonidet P-40 (NP-40)-disrupted virions of all three viruses have only the rA-dA junction. Studies with virus particles which were first disrupted with ether and then treated with NP-40 indicated that the detergent treatment disallowed the formation of the ribopyrimidine-dC internucleotide bond. The same transfers are found with AMV in the presence or absence of actinomycin D, where only single-stranded DNA is formed. This finding is consistent with the notion that virtually all of the significant primers have been recognized. In contrast to mature virions, transfer experiments with ether-disrupted early harvest (5 min) MuLV showed only the rC-dC bond; the rA-dA bond was absent. The short-time harvest contains a significantly higher proportion of infectious virions than 24-h harvests. Also, since the RNA from early harvest virus is appreciably more homogenous than the RNA of mature MuLV, it is concluded that the ribopyrimidine-dC linkage is the more significant initiation event from a biochemical standpoint.