Cell-free synthesis of encephalomyocarditis virus

We developed a system for complete replication of encephalomyocarditis virus (EMCV) in a test tube by using an in vitro translation extract from Krebs-2 cells. Efficient virus synthesis occurred in a narrow range of Mg(2+) and EMCV RNA concentrations. Excess input RNA impaired RNA replication and virus production but not translation. This suggests the existence of a negative-feedback mechanism for regulation of RNA replication by the viral plus-strand RNA or proteins.     

Cell-free synthesis of measles virus proteins.

Polyadenylated mRNA extracted from cytoplasm of measles virus-infected Vero cells was translated in a cell-free system. Three of the polypeptides obtained corresponded to nucleocapsid protein, phosphoprotein, and membrane protein of measles virions. A fourth polypeptide, present in measles virus-infected cells, could be generated by addition of Vero cytoplasmic extract and was identified as a cleavage product of the nucleocapsid protein.     

Proof by synthesis of Tobacco mosaic virus

Background

Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.

Results

RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.

Conclusions

This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.     

Cell-free synthesis of herpes simplex virus proteins.

Polyribosomes isolated from herpes simplex virus type I (HSV-1)-infected cells have been used to program a eucaryotic cell-free translation system. At least 10 HSV-specific polypeptides, with apparent molecular weights of 25,000 to 160,000, are synthesized by wild-type HSV-infected polyribosomes. Polyribosomes prepared from thymidine kinase-negative mutants of HSV direct the synthesis of three putative nonsense termination polypeptides. HSV-specific polypeptides synthesized in vitro are precipitated with antiserum to HSV-infected cell proteins.     

Dynamics of viral RNA synthesis during measles virus infection

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until approximately 24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is approximately 3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5'-end abortive replication fragment RNAs.     

Vaccinia virus produces late mRNAs by discontinuous synthesis

We describe the unusual structure of a vaccinia virus late mRNA. In these molecules, the protein-coding sequences of a major late structural polypeptide are preceded by long leader RNAs, which in some cases are thousands of nucleotides long. These sequences map to different regions of the viral genome and in one instance are separated from the late gene by more than 100 kb of DNA. Moreover, the leader sequences map either upstream or downstream of the late gene, are transcribed from either DNA strand, and are fused to the late gene coding sequence via a poly(A) stretch. This demonstrates that vaccinia virus produces late mRNAs by tagging the protein-coding sequences onto the 3' end of other RNAs.     

Measles virus infection induces chemokine synthesis by neurons

The role that neurons play in the induction of the immune response following CNS viral infection is poorly understood, largely owing to the belief that these cells are immunologically quiescent. In this report, we show that virus infection of neurons results in the synthesis of proinflammatory chemokines, which are early and important mediators of leukocyte recruitment to sites of viral infection. For these studies, a transgenic mouse model of neuron-restricted measles virus (MV) infection was used. Inoculation of immunocompetent and immunodeficient transgenic adult mice resulted in CNS induction of the mRNAs encoding IFN-gamma inducible protein of 10 kD, monokine inducible by gamma and RANTES. Colocalization of chemokine proteins with MV-infected neurons was detected by immunofluorescence in infected brain sections. Both IFN-gamma inducible protein 10 kD and RANTES were also induced in MV-infected primary hippocampal neurons cultured from transgenic embryos, as shown by RNase protection assay, confocal microscopy, and ELISA. Interestingly, neuronal infection with another RNA virus (lymphocytic choriomeningitis virus) was not associated with induction of these chemokines. In immunocompetent mice, chemokine synthesis preceded the infiltration of T lymphocytes, and chemokine ablation by neutralizing Abs resulted in a 20-50% reduction in the number of infiltrating lymphocytes. Collectively, these data indicate that neurons play an important role in the recruitment of a protective antiviral response to the CNS following viral infection, although such a role may be virus type-dependent.     

Inhibitory effect of 1-beta-D-arabinofuranosylthymine on synthesis of Epstein-Barr virus

The effect of 1-beta-D-arabinofuranosylthymine (ara-T) on cell growth and synthesis of Epstein-Barr virus (EBV) in human lymphoblastoid cell lines was determined. The growth of P3HR-1 cells was not inhibited by 1 microgram of the drug per ml; however, infectious virus production was strongly inhibited and was accompanied by decreased expression of early antigen (EA) and viral capsid antigen (VCA). The ability of 12-O-tetradecanoylphorbol-13-acetate or n-butyric acid to induce synthesis of VCA, but not EA, in P3HR-1 cells was inhibited by ara-T. Similarly, VCA synthesis but not EA synthesis was inhibited by ara-T in Jijoye cells superinfected with the P3HR-1 strain of EBV. The results suggest that ara-T has a specific inhibitory action against EBV replication.     

Replacement of murine leukemia virus readthrough mechanism by human immunodeficiency virus frameshift allows synthesis of viral proteins and virus replication

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model.     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.