Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

A system for studying the in vitro replication of the RNA genomes of both wild-type vesicular stomatitis virus (VSV) and its defective interfering particle MS-T has been developed. After lysolecithin treatment of cells infected with VSV or VSV plus MS-T, a cell-free cytoplasmic extract is prepared which will support VSV mRNA synthesis and the synthesis of the 42S wild-type or 19S MS-T genome RNAs. The genome-length RNAs synthesized in vitro are assembled into RNase-resistant nucleocapsids. The level of 42S RNA synthesis in vitro (6-13% of total RNA synthesis) reflects the level of replication in vivo. Although the extracts of VSV-infected cells can also support the synthesis of VSV proteins, RNA replication is not dependent on de novo protein synthesis but utilizes the preformed soluble proteins present in the infected cell at the time the extract is prepared. The initiation of genomic RNA during in vitro replication can be demonstrated because detergent-disrupted, purified MS-T particles will replicate their RNA when added to either a total cytoplasmic extract from VSV-infected cells or the soluble protein fraction derived from such an extract.     

Transcription of the Rous sarcoma virus genome in vitro and in vivo.

RNA-directed DNA synthesis by detergent-disrupted virions of Rous sarcoma virus (RSV) initiates by the covalent attachment of pdA to the 3'-terminal rA of a 4S RNA hydrogen-bonded to the 70S RNA template. This 4S "primer" has structural features of tRNA and can be aminoacylated with methionine. Synthesis and integration of provirus DNA can be monitored in both permissive (duck) and nonpermissive (mouse) cells acutely infected with RSV. The results of these studies, as well as data obtained with RSV-infected mammalian cells which have reverted from a transformed to a pheno-typically normal state, indicate that integration of viral genes into the host chromosome is not sufficient cause for transformation. Pertinent features of virus-specific RNA-directed DNA synthesis in vitro and in vivo are reviewed and compared.     

Rescue of a foreign gene by Sendai virus.

A simple protocol for the rescue of a synthetic genome into a paramyxovirus has been developed. First, a synthetic Sendai virus-like RNA, containing the antisense coding region of the chloramphenicol acetyltransferase gene replacing the coding region of the Sendai virus genome, was transcribed from a cDNA. When introduced into cells that are infected with Sendai virus, this RNA construct was transcribed, replicated, and packaged into infectious virions. The addition of infected cell extract to the RNA prior to transfection markedly enhanced levels of chloramphenicol acetyltransferase expression and rescue. However, this enhancement is not due to encapsidation of the RNA into nucleocapsids as the RNA remains nuclease-sensitive. Uninfected cell extract also enhances expression and rescue efficiency, implying involvement of a cellular factor(s) with the synthetic viral-like RNA construct that allows for enhanced polymerase recognition. This system should allow for the dissection of the various cis-acting RNA signals within the paramyxovirus genome.     

Inhibition of translation by poliovirus: inactivation of a specific initiation factor.

Translation of vesicular stomatitis virus (VSV) mRNA, like host mRNA translation, is inhibited in cells infected with poliovirus. To study the mechanism of poliovirus-induced inhibition of protein synthesis, we prepared extracts from poliovirus-infected and uninfected HeLa cells. Poliovirus mRNA was translated in lysates from both infected and uninfected cells, while VSV mRNA was translated only in the lysate from uninfected cells. Addition of purified translation initiation factors to the extract from infected cells showed that one factor, eIF-4B, could restore VSV mRNA translation in the infected lysate, but did not increase poliovirus mRNA translation. Further experiments involving translation of VSV mRNA in mixed extracts from poliovirus-infected and uninfected cells showed (i) that there was not an excess of an inhibitor of VSV mRNA translation in the infected lysate, but (ii) that an acitivity that caused a slow inactivation of eIF-4B was present in the infected lysate. Inactivation of eIF-4B appears to be the mechanism by which poliovirus infection causes a selective inhibition of translation.     

Multiple forms of sarc gene proteins from Rous sarcoma virus RNA.

In a previous study we were able to identify two proteins of 25,000 and 18,000 daltons that were made from RNA of transforming virions of Rous sarcoma virus (RSV) and that were missing from the translation products of a transformation-defective deletion mutant of RSV. In the present study we have separated RSV virion RNA on sucrose gradients and have determined that the two putative sarc gene products are synthesized as doublets from an mRNA of approximately 18 S. There also appear to be several other sizes of virion mRNA that direct the synthesis of other viral proteins. These data are discussed in terms of the structure of the RSV genome. In addition to the 25,000- and 18,000-dalton doublets, there also is a 60,000-dalton protein whose synthesis is directed by 18S viral RNA from transforming virion of RSV. Peptide mapping has shown that the 60,000- and 25,000-dalton doublet are structurally related. In addition, the use of two-dimensional gel electrophoresis has allowed us to resolve both bands of the 25,000-dalton doublet into several differently charged species.     

Identification and some biochemical properties of the major XBL gene product of bovine leukemia virus.

Using a rabbit antiserum directed against a synthetic oligopeptide whose sequence was deduced from the nucleotide sequence of the XBL gene of bovine leukemia virus, we detected a 38-kDa protein in virus-producing cell lines. In vitro translation of hybrid-selected RNA unequivocally demonstrates that this protein, designated p38(XBL), is indeed encoded by the XBL gene. Unlike the other virus-encoded proteins, however, p38(XBL) resides within the cells without being incorporated into virions. It undergoes no gross post-translational modifications and has a relatively short half-life (5-6 hr) in vivo. Furthermore, cell fractionation combined with pulse-chase experiment reveals that a significant fraction (more than half) of the p38(XBL) localizes to the nucleus of the infected cell after synthesis. We conclude that the XBL gene of bovine leukemia virus is a functional gene encoding a nonvirion protein p38(XBL), which possibly functions within the nucleus of the infected cell to regulate viral or cellular gene expression. p38(XBL) is presumably translated from a doubly spliced, bicistronic mRNA that has the capability to encode another small polypeptide in a different reading frame.     

L612K变异MxA蛋白体外抑制水泡性口膜炎病毒复制的研究

目的研究L612K变异MxA蛋白抑制水泡性口膜炎病毒（VsV）复制活性。方法将野生型MxA蛋白重组表达载体pcDNA3.1MxA（wT）和L612K变异MxA蛋白载体pcDNA3.1-MxA（L612K）分别瞬时转染Wish细胞，24h后VsV感染细胞，感染后48hMTT检测各组细胞增殖数；另取wish细胞转染MxA载体和对照DNA，24h后加入VsV，感染24h后收集细胞，RT-PCR检测VSV mRNA水平，Western blot检测MxA蛋白的表达水平。结果野生型和L612K变异MxA蛋白均在wish细胞有较好表达；MTT检测L612K变异组细胞增殖数明显低于野生型（t=1.13，P〈0.01），RT-PCR检测L612K变异组VsVmRNA水平明显高于野生型组（t=0.13，P〈0.01）。结论L612K变异可能使MxA蛋白降低了对VsV的抑制活性。     

Cell-Free Translation of Messenger RNA of Simian Virus 40: Synthesis of the Major Capsid Protein

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-1. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.     

Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase.

The large gene, L, of vesicular stomatitis virus (VSV), which codes for the multifunctional RNA-dependent RNA polymerase, was assembled from five overlapping cDNA clones. The sequence of the 6.4-kilobase gene of the final construct was identical to the consensus sequence reported earlier. The gene was inserted into the simian virus 40 transient expression vector pJC119. Antibodies directed against synthetic peptides corresponding to the amino and carboxyl termini of the L protein were raised in rabbits. Both antibodies specifically immunostained the cytoplasm of COS cells that had been transfected with the vector DNA. The expressed L protein was immunoprecipitated from cell extracts and it was identical in size to the L protein of the virion (241 kilodaltons). Most importantly, COS cells that expressed the recombinant L protein transcribed, replicated, and consequently complemented and rescued temperature-sensitive RNA polymerase mutants of VSV at the nonpermissive temperature. The kinetics of virus release were similar to those of a wild-type VSV infection. We conclude that the recombinant RNA polymerase protein L is indistinguishable in its size and its functions from the VSV polymerase.     

tRNA elements mediate the assembly of an icosahedral RNA virus.

tRNAs, the adapter molecules in protein synthesis, also serve as metabolic cofactors and as primers for viral RNA-directed DNA synthesis. The genomic and subgenomic RNAs of some plant viruses have a 3'-terminal tRNA-like structure (TLS) that can accept a specific amino acid and serve as a site for initiation of replication and as a simple telomere. We report a previously undescribed role for the TLS of brome mosaic virus (BMV), and potentially for cellular tRNA, in mediating the assembly of its icosahedral virions. BMV genomic RNAs and subgenomic RNA lacking the TLS failed to assemble into virions when incubated with purified BMV coat protein. Assembly was restored by addition of a 201-nt RNA containing the BMV TLS. TLSs from two other plant viruses as well as tRNAs from wheat germ and yeast were similarly active in the BMV virion assembly reaction, but ribosomal RNA and polyadenylate did not facilitate assembly. Surprisingly, virions assembled from TLS-less BMV RNA in the presence of tRNAs or TLS-containing short RNA did not incorporate the latter molecules. Consistent with a critical role for the BMV TLS in virion assembly, mutations in the BMV genomic RNAs that were designed to disrupt the folding of the TLS also abolished virion assembly. We discuss the likely roles of the TLS in early stages of virion assembly.     

A human monoclonal antibody that recognizes viral polypeptides and in vitro translation products of the genome of the hepatitis D virus

Peripheral blood lymphocytes from a patient chronically infected with hepatitis D virus (HDV) were immortalized by Epstein-Barr virus transformation. Two stable monoclonal cell lines, derived from the same parent culture, were established and produced antibodies of the IgG isotype that were specific for the hepatitis delta antigen (HDAg). Both monoclonal antibodies (MAbs) recognized the major HDAg polypeptides of 24 kilodaltons and 27 kilodaltons that were previously detected by polyclonal antibodies to HDAg in both liver and serum from HDV-infected humans, chimpanzees, and woodchucks. This result indicates that the major polypeptides of HDAg share common epitopes. The MAbs also reacted with minor polypeptides of lower molecular weight, which were present in infected liver. In vitro translation products of HDV-specific RNA from infected liver were also detected by the MAbs; these polypeptides were 24 kilodaltons and 27 kilodaltons, respectively, and comigrated with liver- or serum-derived HDAg. In contrast, HDV RNA isolated from virions in serum was not translated into HDAg polypeptides in the in vitro system.     

In vitro assembly of the nonglycosylated membrane protein (M) of Sendai virus.

The nonglycosylated membrane protein (M) of Sendai virus was purified from virions and conditions were found under which the protein assembled in vitro into three types of ordered structures: narrow tubes, wide tubes, and sheets. These structures were examined by high resolution electron microscopy by using negative staining and metal shadowing techniques. The tubes and sheets are formed from strands 7.2 nm wide that are composed of annular subunits. The wide tubes appear to be formed by the rolling of a sheet into a cylinder in which the 7.2-nm strands are inclined with a pitch of 26-33 degrees and have a left-handed orientation. In addition to the strong reflections corresponding to the 7.2-nm spacings generated by the strands, optical diffraction patterns also showed weak reflections that could be indexed on a lattice corresponding to real-space lattice constants of 7.6 nm and 5.3 nm, with an included angle of 71 degrees. The dimensions and arrangements of these structures formed in vitro are strikingly similar to those of ordered arrays of particles found by others to be associated with the inner surface of the plasma membrane of infected cells. The results support the concept that ordered arrays of M protein, similar to those assembled in vitro, are involved in the assembly of the virus particle by budding from the cell membrane and that they provide specific recognition sites for the viral nucleocapsid at the cytoplasmic surface of the plasma membrane.     

In vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded RNA virus.

A system is described for assembling infectious bacteriophage phi 6 nucleocapsids in vitro. Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments. The resulting filled particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8. The infected spheroplasts yielded infectious virions. A modified cDNA-derived RNA segment was inserted into virions by this method. The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA. These findings support the contention that the preformed procapsids are the "machine" that replicates the phi 6 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.     

Proteins of purified Epstein-Barr virus

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.