A cis-acting element within the hepatitis A virus 5'-non-coding region required for in vitro translation.

Every picornavirus studied thus far has a sequence within the 5'-non-coding region that is required for internal ribosome binding and translation of the polyprotein. In an attempt to identify this region in hepatitis A virus we constructed a truncated hepatitis A virus (HAV) cDNA clone that contains the entire 736 bp 5' non-coding region (5'-NCR) and 754 base pairs of the viral capsid coding region (P1) under control of the SP6 promoter. In vitro transcription and translation of this transcript in a rabbit reticulocyte lysate yielded a protein product of about 29 kDa as analyzed by autoradiography following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A series of mutations of this construct have defined a minimal sequence between bases 347 and 734 in the 5'-NCR that is required for efficient in vitro translation. The deleted constructs (D 523-734 and D 632-734) showed a reduced ability to translate in the rabbit reticulocyte lysate system in comparison with the full-length 5'-NCR construct, pH1489. The translation of these deleted constructs was artificially restored by the addition of a 5'-terminal methylated cap structure, m7GpppG, to the RNA. This increase in translational efficiency could be competed away with cap analog (m7GDP) thus indicating that this region is required for cap-independent internal ribosome binding for HAV translation.

Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit.

The influenza A virus RNA-dependent RNA polymerase catalyzes several reactions in transcription and replication of the genome RNA. The first step in viral mRNA synthesis is the recognition of the 5' end cap structure of host cell hnRNA and the cleavage of the RNA substrate between 10 and 14 nucleotides from the 5' end to generate capped primers for initiation of transcription of virus-specific mRNAs. This report describes the use of an in vitro UV crosslinking and protein renaturation assay to identify the polymerase subunits which interact with the 5' end cap structure of an artificial RNA substrate. Our results showed, for the first time, that purified polymerase subunit PB2 expressed by recombinant baculovirus in insect cells possessed cap-binding activity by itself after renaturation by Escherichia coli thioredoxin, whereas cleavage of the artificial capped substrate required the holoenzyme expressed in insect cells triply-infected with baculovirus containing all three polypeptide components, PB1, PB2, and PA. Purified polyclonal anti-PB2 IgG inhibited the binding activity; anti-PB1 and anti-PA IgGs did not.

Detection of hepatitis A virus RNA and capsid antigen in individual cells.

The replication of hepatitis A virus (HAV) RNA and the production of HAV VP1 protein were examined in cultures of BS-C-1 cells under one-step growth conditions by in situ hybridization and immunofluorescence. Individual cells that had undergone active viral RNA replication were detectable at 24 h post-infection. During subsequent days, increasing numbers of cells began replicating viral RNA, so that by seven days post-infection, all cells had accumulated significant amounts of viral RNA. The results show that the protracted replication cycle of HAV in cultured cells represents a slow recruitment of infected cells into a replication mode, rather than an inherently slow virus reproduction in all cells. With the reagents utilized in this study, nucleic acid hybridization was more sensitive than antigen detection by immunofluorescence or immunoblot analysis.

Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro.

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.

Expression of hepatitis A virus capsid sequences in insect cells.

A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.

Influenza A virus NP protein expressed in insect cells by a recombinant baculovirus is associated with a protein kinase activity and possesses single-stranded RNA binding activity.

Influenza A virus NP protein, the phosphoprotein associated with viral RNA in ribonucleoprotein (RNP) complexes, has been expressed at high levels (approximately 100 mg/liter cells) in insect (Sf9) cells by a baculovirus recombinant, and was localized almost entirely in the nuclei of these cells. NP was purified by immuno-affinity chromatography, and purified NP was shown to autophosphorylate and to phosphorylate casein in a cAMP-independent reaction. Furthermore, purified NP was able to bind to ssRNA as demonstrated by a mobility shift of ssRNA in non-denaturing gels. The binding of NP to ssRNA caused a diminution of its kinase activity in proportion to binding.

Efficient elution of purified proteins from polyvinylidene difluoride membranes (Immobilon) after transfer from SDS-PAGE and their use as immunogens.

Following separation of proteins by SDS-PAGE, they are electroblotted onto polyvinylidene difluoride membranes (Immobilon). Protein bands of interest are excised, and the proteins are eluted from the membrane with detergent-containing buffers at pH 9.5. The method routinely yields recovery of 70-90%, and this is independent of protein molecular weight.

Efficient Elution of Purified Proteins from Polyvinylidene Difluoride Membranes (Immobilon) After Transfer from SDS-PAGE and Their Use as Immunogens.

The great analytical power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) makes it one of the most effective tools of protein chemistry and molecular biology. In the past, there have been many attempts to convert the technique from analytical to preparative scale because, by SDS-PAGE, one can resolve more than one hundred protein species in 5-6 h. The number of papers that describe preparative elution from polyacrylamide gels is immense (for example, see refs. 1-5), In spite of the numerous variations in the procedure of elution, none of the available methods is entirely satisfactory. Some of the methods are very laborious, and others lead to loss of resolution or poor recovery.

Fluorescent protein staining on nitrocellulose with subsequent immunodetection of antigen.

The transfer of proteins from gels to nitrocellulose or other immobilizing matrices has become increasingly popular as a powerful tool for the subsequent analysis of proteins. Most frequently, the blotted proteins are analyzed for their antigenic properties by Western blotting.

Efficient Elution of Purified Proteins from Polyvinylidene Difluoride Membranes (Immobilon) After Transfer from SDS-PAGE and Their Use as Immunogens.

The great analytical power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) makes it one of the most effective tools of protein chemistry and molecular biology. In the past, there have been many attempts to convert the technique from analytical to preparative scale because, by SDS-PAGE, one can resolve more than one hundred protein species in 5-6 h. The number of papers that describe preparative elution from polyacrylamide gels is immense (for example, see refs. 1-5), In spite of the numerous variations in the procedure of elution, none of the available methods is entirely satisfactory. Some of the methods are very laborious, and others lead to loss of resolution or poor recovery.

Fluorescent protein staining on nitrocellulose with subsequent immunodetection of antigen.

The transfer of proteins from gels to nitrocellulose or other immobilizing matrices has become increasingly popular as a powerful tool for the subsequent analysis of proteins. Most frequently, the blotted proteins are analyzed for their antigenic properties by Western blotting.

Identification of a trans-acting activity from liver that stimulates hepatitis A virus translation in vitro.

Hepatitis A virus (HAV), a picornavirus, is the causative agent of infectious hepatitis, generally a self-limiting disease of the liver. Recently, sequences within the 5' noncoding region that affect the translation of the viral genome have been identified using in vitro systems. In this report we demonstrate that extracts prepared from mouse liver cytoplasm specifically stimulate HAV RNA translation in a rabbit reticulocyte lysate in vitro. This activity appears to act specifically on HAV sequences and is not found in other mouse tissue and several cell lines of tissue culture origin.

Electron microscopy of vesicular stomatitis virus replicative ribonucleoproteins.

The objective of this investigation was to examine by electron microscopy the replicative ribonucleoprotein (RNP) structures synthesized in vesicular stomatitis virus-infected HeLa cells. Pulse-labeled in vivo products of vesicular stomatitis virus replication and transcription can be separated by centrifugation in Renografin gradients. Transcription complexes are dissociated, allowing nascent messenger RNPs to remain at the top of the gradient, whereas RNPs biochemically consistent with replication complexes sediment to the middle of the gradient. Examination of these structures by electron microscopy revealed that all exist as coiled or helical RNPs having dimensions of approximately 20 by 700 nm. These structures can be further subdivided into three major morphological classes: (i) linear forms (20 by 769 +/- 158 nm), which have both ends free; (ii) circular forms (20 by 679 +/- 95 nm), which appear to have both ends joined; and (iii) complex forms, which include those structures which are branched replicative complexes as well as those which are random. To distinguish random complexes and possible transcriptive complex contaminants from replicative complexes, it was necessary to uncoil the RNP structures with EDTA so that length measurements could be made relating the nascent strand length to its position on the template. After EDTA treatment, the linear RNPs uncoiled (10 by 4,035 +/- 3,802 nm), and the circular morphology virtually disappeared. However, a new form appeared which was one-half the length and double the width (20 by 2,103 +/- 306 nm) of full-length RNPs and contained a loop at one end and two free ends at the other (alpha-form RNP). The distribution and length analysis of these structures, plus and minus EDTA, suggest that the alpha-form RNPs arise by EDTA-induced uncoiling of circular forms held together at the ends. Close scrutiny of uncoiled complex RNPs revealed no single-strand RNP templates with single-strand nascents. However, several complexes were observed which appeared to contain alpha-form templates with single-strand nascent RNPs. Length measurements suggest these complexes are neither random nor transcriptive, but are replicative. These experiments suggest that replication may, in part, occur on circular coiled RNP templates.

Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera.

A sensitive staining method for protein blots on nitrocellulose is described. It is based on the coupling of a fluorochrome, dichlorotriazynylaminofluorescein, to protein which yields products colorless in visible light but colored when protein blots are illuminated with long-range ultraviolet light. The coupling of a fluorochrome does not affect the antigenic properties of proteins and the stained blots can be subsequently probed with antisera. Thus, the method allows for the unambiguous identification of antigenic proteins transferred to nitrocellulose from sodium dodecyl sulfate-polyacrylamide gels.

Newcastle disease virus infection of L cells.

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.

Phosphorylation of vesicular stomatitis virus in vivo and in vitro.

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.