Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.     

Defective interfering viral particles in acute dengue infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.     

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.     

Preliminary physical mapping of RNA-RNA linkages in the genomic RNA of Moloney murine leukemia virus

Retrovirus particles contain two copies of their genomic RNA, held together in a dimer by linkages which presumably consist of a limited number of base pairs. In an effort to localize these linkages, we digested deproteinized RNA from Moloney murine leukemia virus (MLV) particles with RNase H in the presence of oligodeoxynucleotides complementary to specific sites in viral RNA. The cleaved RNAs were then characterized by nondenaturing gel electrophoresis. We found that fragments composed of nucleotides 1 to 754 were dimeric, with a linkage as thermostable as that between dimers of intact genomic RNA. In contrast, there was no stable linkage between fragments consisting of nucleotides 755 to 8332. Thus, the most stable linkage between monomers is on the 5' side of nucleotide 754. This conclusion is in agreement with earlier electron microscopic analyses of partially denatured viral RNAs and with our study (C. S. Hibbert, J. Mirro, and A. Rein, J. Virol. 78:10927-10938, 2004) of encapsidated nonviral mRNAs containing inserts of viral sequence. We obtained similar results with RNAs from immature MLV particles, in which the dimeric linkage is different from that in mature particles and has not previously been localized. The 5' and 3' fragments of cleaved RNA are all held together by thermolabile linkages, indicating the presence of tethering interactions between bases 5' and bases 3' of the cleavage site. When RNAs from mature particles were cleaved at nucleotide 1201, we detected tethering interactions spanning the cleavage site which are intramonomeric and are as strong as the most stable linkage between the monomers.     

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.     

Crystallization of bacteriophage MS2.

Crystals of bacteriophage MS2 have been obtained by slowly cooling a 1% virus solution from 23 degrees C to 0 degrees C in the presence of poly(ethylene glycol) 6000. The crystals were colorless, needle-like, anisotropic and very fragile. Electron microscopic observation of the crystals revealed a two-dimensional lattice of particles with RNA phage morphology and dimensions. Preliminary X-ray examination of the crystals confirmed their viral nature.     

Purification of murine oncornaviral phosphoproteins using alkyl agarose derivatives..

Methods for the purification of both murine mammary tumor (type B) and murine leukemia (type C) oncornaviral phosphoproteins are described, in which chromatography on alkyl-agarose derivatives is used as the primary fractionation step. Gel filtration or ion exchange chromatography on phosphocellulose was the only subsequent step required for the purification of the type B and type C viral proteins, respectively. The two-step protocols also resulted in the co-purification of a low molecular weight core protein from each virus. Recoveries of the viral proteins purified by this method, based on per cent contribution of individual polypeptides to total virion proteins, were 70% or greater. Radioimmunocompetition analysis of the purified murine mammary tumor virus major core protein as well as analysis of the RNA binding properties of purified low molecular weight type C virus proteins suggests that neither antigenic reactivity nor specific RNA binding characteristics are altered by the purification protocols. The availability of these procedures should aid studies on the possible function and immunochemical properties of the native murine oncornaviral phosphoproteins and may also be extended to the purification of other oncornaviral proteins.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus.

Phosphorylation of core particles derived either from hepatitis B viruses or from livers of hepatitis B-infected individuals has been long recognized, but the nature and function of the phosphorylating enzyme remained unknown. By immunoblotting with a monoclonal antibody, we have now detected protein kinase C within the liver-derived core particles. To study the significance of the encapsidated protein kinase C for the viral life cycle, we established an in vitro assembly system consisting of Escherichia coli-expressed core protein, protein kinase C, and in vitro-synthesized hepatitis B virus RNA. Phosphorylation of the core protein led to a reduced RNA encapsidation capacity of the core particles. Furthermore, RNA and protein kinase C competed for their target sequence, which is the carboxy-terminal arginine-rich domain of the core protein. This finding implies that phosphorylation of the nucleic acid binding site in the core protein occurs within the particles after encapsidation of protein kinase C, pregenomic RNA, and viral polymerase at a later step during viral genome maturation.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures

[3H]uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA. Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells. Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions. Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells. Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus. The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus. The ratio could be shifted by passaging virus from one cell type to the other. Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells. Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity. However, passage in C3H/RV cells resulted in the complete loss of infectious virus. Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles. A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other. The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells. The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed.     

The genome-associated,specific RNA binding proteins of avian and mammalian type C viruses

A structural protein purified from the Rous sarcoma virus (RSV) can specifically bind in vitro to purified avian, but not mammalian, type C viral RNA. Following ultraviolet irradiation of viral particles under conditions which stabilize the polyploid 70S viral RNA, the same polypeptide can be directly purified from the RSV genome. Based on its electrophoretic mobility in polyacrylamide gels containing sodium dodecylsulfate, the RNA binding protein has been identified as the major phosphoprotein (p19) of avian type C viruses. Similar experiments show that the major phosphoproteins of mammalian type C viruses (p12 for murine viruses and p16 for endogenous primate viruses) are also the specific RNA binding proteins and, similarly, are found closely associated with the 70S RNA genomes in the intact viral particles.     

Purification and analysis of RNA from paraffin-embedded tissues

We have developed methods for the purification and analysis of RNA from formalin-fixed and paraffin-embedded tissue. The methods allow retrospective analysis of gene expression or viral infection. RNA extraction uses prolonged solubilization of tissue with detergent and protease in the presence of high concentrations of a ribonuclease inhibitor. The purified RNA is moderately degraded but its hybridization behavior is essentially unaffected. We were able to quantify specific mRNAs by dot-blot hybridization.     

RNA Control of HIV-1 Particle Size Polydispersity

HIV-1, an enveloped RNA virus, produces viral particles that are known to be much more heterogeneous in size than is typical of non-enveloped viruses. We present here a novel strategy to study HIV-1 Viral Like Particles (VLP) assembly by measuring the size distribution of these purified VLPs and subsequent viral cores thanks to Atomic Force Microscopy imaging and statistical analysis. This strategy allowed us to identify whether the presence of viral RNA acts as a modulator for VLPs and cores size heterogeneity in a large population of particles. These results are analyzed in the light of a recently proposed statistical physics model for the self-assembly process. In particular, our results reveal that the modulation of size distribution by the presence of viral RNA is qualitatively reproduced, suggesting therefore an entropic origin for the modulation of RNA uptake by the nascent VLP.