Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.     

Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells.

Primary or continuous lines of mouse cells (3T3) are nonpermissive for simian virus 40 (SV40). Abortively infected cells synthesize tumor antigen (T antigen but not viral DNA and virus capsid protein (V antigen). V antigen, however, was obtained when SV40 DNA was injected into 3T3 cells. This late gene expression also appears to be correlated with the quantity of injected DNA molecules per 3T3 cell. T antigen formation can be detected after microinjection of only 1 to 2 DNA molecules, but the intensity of intranuclear T antigen fluorescence is significantly brighter with injection of higher concentrations of viral DNA. In permissive cells (TC7), early and late SV40 gene expression is directly related to the number of injected molecules. Microinjection of 1DNA molecule induced T and V antigen formation with the same efficiency as microinjection of 2,000 to 4,000 molecules. The question of weather late SV40 gene expression is directly related to the quantity of an early virus-specific product was approached by microinjection of early SV40 complementary RNA together with small amounts of viral DNA. V antigen was obtained in a high proportion of recipient 3T3 cells at conditions where microinjection of viral DNA alone induced T but not V antigen synthesis.     

Effect of a tsA mutation of simian virus 40 late gene expression: variations between host cell lines

Infection of AGMK or CV-1 cells by the early simian virus 40 mutant tsA58 at the permissive temperature (32 degrees C) followed by a shift to the nonpermissive temperature (41 degrees C) caused a substantial decrease in the levels of late viral RNA in the cytoplasm of AGMK cells but not CV-1 cells. At the translational level, this depression of late viral RNA levels was reflected by a decrease in late viral protein synthesis. Thus, in AGMK cells, an early region gene product (presumably large T-antigen) appeared to be continuously required for efficient expression of the late viral genes. In contrast, late simian virus 40 gene expression, once it is initiated in CV-1 cells, continued efficiently regardless of the tsA mutation. The difference in expression of the late simian virus 40 genes in these tsA mutant-infected monkey kidney cell lines may reflect a difference in host cell proteins which regulate viral gene expression in conjunction with early viral proteins.     

Development of E.coli virus T1: the pattern of gene expression.

Summary T1 infected bacteria exhibit a distinct pattern of gene expression. The control of this expression is accessible to biochemical analysis. T1 induces the synthesis of 31 proteins inE. coli. The virion contains 15 proteins. By means of T1 amber mutants, 10 gene products have been assigned to specific T1 genes. Three classes of T1 proteins are defined by the kinetics of their syntheses: early, early-late and late proteins. The regulation of protein synthesis involves at least three mechanisms: for cessation of host gene expression, for discontinuation of the early class during the late phase and for induction of the late T1 proteins. The positive control of late gene expression is not coupled to replication. The host RNA-polymerase transcribes the viral genome throughout the infectious cycle. No virus coded RNA-polymerase is induced.     

Analysis of expression of adenovirus DNA (fragments) by microinjection in Xenopus oocytes. Independent synthesis of minor early region 2 proteins

Injection of whole adenovirus DNA into Xenopus oocytes results in the synthesis of large amounts of the early region 2A DNA-binding protein (E2A-DBP) and smaller amounts of polypeptide IX. The lack of synthesis of any functional messenger RNAs transcribed from the major late promotor at 16.3 map units is remarkable. Cleavage of the adenovirus DNA outside the E2A gene proper by restriction enzymes decreases synthesis of the DBP to about 10% of the amount produced after injection of intact DNA. On the other hand, presence of the terminal (Bellett) protein on the injected template enhances DBP synthesis considerably. Experiments with injected DNA restriction fragments, as well as reconstructed genes cloned into pBR322, indicate that efficient synthesis of DBP in oocytes requires the presence of either or both of the two main promoters from which the E2A gene is transcribed plus an intact 3' end of the gene. In the absence of any known promotor, 100-fold lower amounts of otherwise normal DBP are produced. Unlike in a regular infection, synthesis of DBP in oocytes does not require the product of the E1A gene. The same series of experiments also demonstrates that the DBP, a phosphoprotein, is the substrate of a cellular rather than a virus-encoded protein kinase. Two minor E2A proteins, although colinear with the major DBP, are synthesized independently. Synthesis of a 44,000 Mr protein, probably corresponding to the carboxy-terminal 360 amino acid residues of the DBP, is not decreased after injection of "promotorless" E2A genes. Unlike the 44,000 Mr protein, production of a 67,000 Mr protein (carboxy-terminal 483 amino acid residues) by one DNA-construct is probably directed by a T-A-T-A-A-A-T-A sequence in the vector DNA.     

Replication of poliovirus in Xenopus oocytes requires two human factors.

We described a novel system to study poliovirus replication in Xenopus oocytes. Poliovirus RNA microinjected into Xenopus oocyte initiates a complete cycle of viral replication, yielding a high level of infectious viruses. Two distinct HeLa cell activities are required, one involved in initiation of translation and the other in RNA synthesis. The translation factor is a large cytoplasmic protein or complex, which is specifically used for initiation of poliovirus translation. The replication factor is required at early stages of RNA synthesis. Formation of infectious poliovirus is highly temperature dependent. At temperatures below 27 degrees C, capsid assembly appears to be impaired. The oocyte system described here could be useful in identifying and characterizing viral and cellular factors involved in virus replication.     

RNA interference as a tool to study gene function in bovine oocytes

RNA interference (RNAi) has become a well-established technique to study gene function in several species. Our objective was to develop a RNAi approach to study gene function in bovine oocytes. In the first experiment, three different treatments including a 20 min exposure to cytochalasin B, a 6 hr maturation in cycloheximide, and a combination of these two treatments were tested to improve oocyte survival following microinjection. The cycloheximide/cytochalasin B treatment greatly increased (P<0.02) the survival rate of the microinjected oocytes. In the second experiment, we assessed the effect of both cyclin B1 and GFP dsRNA on cyclin B1 mRNA and protein expression. The injection of cyclin B1 dsRNA resulted in a decrease in cyclin B1 mRNA and protein, while the cyclin B2 mRNA remained unaffected. Furthermore, the injection of GFP dsRNA did not interfere with cyclin B1 mRNA or protein nor with the ability of the oocyte to mature properly. In addition, the lack of cyclin B1 in the oocyte led to activation in 10% of the oocytes as evidenced by the presence of a pronucleus. However, the use of an additional 10 hr of maturation in the presence of 6-dimethylaminopurine (6-DMAP) prevented germinal vesicle breakdown and allowed a longer exposure to dsRNA. This procedure increased the percentage of activated oocytes to 33% and is likely to result from an increased length of time for dsRNA processing and for degradation of the cyclin B1 mRNA to occur. In conclusion, RNAi represents a useful technique to study gene function in the bovine oocyte.     

Redundant control of adenovirus late gene expression by early region 4.

A series of human adenovirus type 5 derivatives carrying deletion mutations in early region 4 (E4) were constructed and characterized with respect to viral late protein synthesis, viral cytoplasmic late message accumulation, viral DNA accumulation, and plaquing ability. Viral late protein synthesis was essentially normal in cells infected by mutants expected to produce either the E4 open reading frame (ORF) 3 product or the E4 ORF 6 product. In cells infected by mutants lacking both ORF 3 and ORF 6, late protein synthesis was dramatically reduced. The basis for this reduction appears to be a concomitant reduction in cytoplasmic late message levels. Our results suggest that the products of ORFs 3 and 6 are redundant, since they are individually able to satisfy the requirement for E4 in late gene expression. Two of the mutants examined were defective for viral late protein synthesis but showed no measurable defect in viral DNA accumulation. The defect in late gene expression is not, therefore, a reflection of a primary defect in viral DNA synthesis. Finally, mutants expected to express ORF 3 or ORF 6 formed plaques with normal or only modestly reduced efficiency, whereas mutants expected to express neither ORF formed plaques with an efficiency less than 10(-6) that of wild-type virus. Thus, plaque-forming ability reflected late protein synthetic ability, suggesting that among these mutants late protein synthetic proficiency is the principle determinant of plaquing efficiency.