RNA degradation induced by the herpes simplex virus vhs protein proceeds 5' to 3' in vitro

The herpes simplex virus virion host shutoff protein (vhs) is an mRNA-specific RNase that contributes to shutoff of host protein synthesis. We show here that vhs-induced mRNA decay proceeds 5' to 3' in an in vitro assay system derived from rabbit reticulocyte lysate.     

Avian erythroblastosis virus produces two mRNA''s.

We analyzed the viral mRNA's present in fibroblast nonproducer clones transformed by avian erythroblastosis virus. Two size classes of mRNA (28 to 30S and 22 to 24S) were identified by solution hybridization with both complementary DNA strong stop and complementary DNA made against the unique sequences of avian erythroblastosis virus. Based upon the kinetics of hybridization with complementary DNA made against the unique sequences of avian erythroblastosis virus, we estimated that there were 400 to 500 copies of the 28 to 30S RNA per cell and 200 to 250 copies of the 22 to 24S RNA per cell. Both RNA species were packaged in the virion. In vitro translation of the 28 to 30S virion RNA yielded a 75,000-dalton protein which was the 75,000-dalton gag-related polyprotein found in avian erythroblastosis virus-transformed cells. In vitro translation of the 22 to 24S virion RNA yielded two proteins (46,000 and 48,000 daltons). This indicates that there may be two genes in avian erythroblastosis virus, one coding for the 75,000-dalton gag-related polyprotein and the second coding for the 46,000- or 48,000-dalton protein or both.     

An efficient ligation method in the making of an in vitro virus for in vitro protein evolution

The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion(=viral particle)”(mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated. Published: October 28, 2002     

Identification of a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.     

Polyoma virus has three late mRNA''s: one for each virion protein.

Polyoma virus mRNA, isolated from the cytoplasm of 3T6 cells late after infection and purified by hybridization to HpaII fragment 3 of polyoma virus DNA, was separated on 50% formamide-containing sucrose density gradients, and the fractionated RNA was recovered and translated in vitro. Analysis of the cell-free products showed that the minor virion protein VP3 was synthesized from an mRNA sedimenting at approximately 18S betweeen the 19S VP2 mRN and the 16S VP1 mRNA. Other experiments showed that the VP2 and VP3 can be labeled with formyl methionine from initiator tRNA. We conclude that there are three late polyoma virus mRNA's, each directing the synthesis of only one viral capsid protein.     

The virion host shutoff protein of herpes simplex virus type 1: messenger ribonucleolytic activity in vitro.

Shortly after tissue culture cells are infected with herpes simplex virus (HSV) type 1 or 2, the rate of host protein synthesis decreases 5- to 10-fold and most host mRNAs are degraded. mRNA destabilization is triggered by the virion host shutoff (vhs) protein, a virus encoded, 58-kDa protein located in the virion tegument. To determine whether it can function as a messenger RNase (mRNase), the capacity of vhs protein to degrade RNA in vitro in absence of host cell components was assessed. Two sources of vhs protein were used in these assays: crude extract from virions or protein translated in a reticulocyte-free system. In each case, wild-type but not mutant vhs protein degraded various RNA substrates. Preincubation with anti-vhs antibody blocked RNase activity. These studies do not prove that vhs protein on its own is an mRNase but do demonstrate that the protein, either on its own or in conjunction with another factor(s), has the biochemical property of an mRNase, consistent with its role in infected cells.     

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.     

Analysis of herpes simplex virus-induced mRNA destabilizing activity using an in vitro mRNA decay system.

Most host mRNAs are degraded soon after infection of cells with herpes simplex virus type 1 (HSV-1). This early shutoff or early destabilization response is induced by a virion component, the virion host shutoff (vhs) protein. HSV-1 mutants, vhs1 and vhs-delta Sma, which produce defective or inactive vhs protein, fail to induce early shutoff. We have used an in vitro mRNA decay system to analyze the destabilization process. Polysomes from uninfected human erythroleukemia cells, used as a source of target mRNAs, were mixed with polysomes or with post-polysomal supernatant (S130) from HSV-1- or mock-infected murine erythroleukemia cells. Normally stable gamma-globin mRNA was destabilized by approximately 15-fold with S130 from wild-type virus-infected cells but was not destabilized with S130 from mock-infected cells or from cells infected with either of the two HSV mutants. The virus-induced destabilizing activity had no significant effect on the in vitro half-lives of two normally unstable mRNAs, histone and c-myc. No destabilizing activity was detected in polysomes from infected cells. We conclude that a virus-induced destabilizer activity can function in vitro, is located in the S130 of infected cells, and accelerates the decay rates of some, but not all, polysome-associated host mRNAs.     

Synthesis of polyoma proteins in vitro

Polyoma virus DNA can be transcribed and translated in an extract of Escherichia coli. Proteolytic digests of the product contain peptides that correspond to peptides from the virion. One of the in vitro synthesized polypeptides corresponds in size to the major virion protein but most of the product is smaller in size than this protein. Although most of the tryptic peptides corresponding to the major virion protein can be detected in the in vitro synthesized polypeptides, all of them also gave rise to other, non-matching, peptides. Much of the in vitro product therefore appears to correspond to incomplete molecules of the major virion protein.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.     

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line

Parvoviruses are small, 260-A-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.     

Antibody-mediated uncoating of adenovirus in vitro

In a virus destabilization assay in vitro it was demonstrated that exposure of adenovirus to proteins will non-specifically protect the virus from being uncoated following transfer to low pH and hypotonic conditions. Such uncoating was also fully inhibited upon pretreatment of virus with 0.05% of the non-ionic detergent polyoxyethylenesorbitan monolaurate (Tween 20). However, in the presence of low concentrations of Tween 20 it was shown that monospecific immunoglobulins, directed against the fiber antigen and polyspecific antibodies produced in response to intact virions, were able to overcome the detergent-protecting effect of uncoating. Immunoglobulins directed towards the remaining outer-capsid components, the hexon, the penton base and the protein IIIa, revealed no such effects. The antifiber-mediated uncoating was paralleled by an aggregation of the virions. The data suggest that the virion-stabilizing effect of salt is enhanced by the hydrophobic action of a non-ionic detergent. Under these conditions the interaction between antifiber antibodies and fibers of the virion will trigger a destabilization of the virion upon transfer to low pH and hypotonic conditions.