Similar regulation of the synthesis of adenovirus fiber and of simian virus 40-specific proteins encoded by the helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del.

Human adenoviruses fail to multiply effectively in monkey cells. The block to the replication of these viruses can be overcome by coinfection with simian virus 40 (SV40) or when part of the SV40 genome is integrated into and expressed as part of the adenovirus type 2 (Ad2) genome, as occurs in several Ad2+SV40 hybrid viruses, such as Ad2+ND1, Ad2+ND2, and Ad2+ND4. The SV40 helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del were analyzed to determine why they are unable to grow efficiently in monkey cells even though they contain the appropriate SV40 genetic information. Characterization of the Ad2+ND5-SV40-specific 42,000-molecular-weight (42K) protein revealed that this protein is closely related, but not identical, to the SV40-specific 42K protein of the SV40 helper-competent Ad2+ND2 hybrid virus. Although the minor differences between these proteins may be sufficient to account for the poor growth of Ad2+ND5 in monkey cells, the most striking difference between helper-competent Ad2+ND2 and helper-defective Ad2+ND5 is in the production of the SV40-specific protein after infection of monkey cells. Whereas synthesis of the SV40-specific proteins of Ad2+ND2 is very similar in human and in monkey cells, production of the 42K protein of Ad2+ND5 is dramatically reduced in monkey cells compared with human cells. Similarly, the synthesis of the SV40-specific proteins of Ad2+ND4del is markedly reduced in monkey cells. Thus, it is likely that both Ad2+ND5 and Ad2+ND4del are helper defective because of a block in the production of their SV40-specific proteins rather than because their SV40-specific proteins are nonfunctional. This block, like the block to adenovirus fiber synthesis, is overcome by coinfection with SV40, with helper-competent hybrid viruses, or with host range mutants of adenoviruses. This suggests that the synthesis of fiber and the synthesis of SV40-specific proteins are similarly regulated in Ad2+SV40 hybrid viruses.     

Conditional lethal mutants of adenovirus type 2-simian virus 40 hybrids. II. Ad2+ND1 host-range mutants that synthesize fragments of the Ad2+ND1 30K protein.

Adenovirus type 2 (Ad2) grows 1,000 times less well in monkey cells than in human cells. This defect can be overcome, not only upon co-infection of cells with simian virus 40 (SV40), but also when the relevant part of the SV40 genome is integrated into the adenovirus genome to form an adenovirus-SV40 hybrid virus. We have used the nondefective Ad2-SV40 hybrid virus Ad2+ND1, which contains an insertion of 17% of the SV40 genome, to isolate host-range mutants which are defective in growth on monkey cells although they grow normally on human cells. Like Ad2, these mutants are defective in the synthesis of late proteins in monkey cells. A 30,000-molecular-weight protein (30K), unique to Ad2+ND1-infected cells, can be synthesized in vitro, using Ad2+ND1 mRNA that contains SV40 sequences. 30K is not seen in cells infected with those host-range mutants that are most defective in growth on monkey cells, and translation in vitro of SV40-specific mRNA from these cells produces new unique polypeptides, instead of 30K. Genetic and biochemical analyses indicate that these mutants carry point mutations rather than deletions.     

Characterization of the translational defect to fiber synthesis in monkey cells abortively infected with human adenovirus: role of ancillary leaders

Synthesis of the fiber protein of human adenovirus serotype 2 (Ad2) is 100-fold lower in abortively infected monkey cells, compared with productive infections, despite only a 5- to 10-fold reduction in fiber mRNA levels. Previously Anderson and Klessig (Proc. Natl. Acad Sci. USA 81:4023-4027, 1984) demonstrated a direct correlation between the productive nature of the infection, efficient synthesis of the fiber protein in vivo, and the presence of the x or y ancillary leaders on 10 to 25% of fiber messages. To determine at what level in translation these leaders might be important, the relative rate of initiation and elongation of each class of fiber message was assessed. The presence of the y ancillary leader in productively infected cells increased the rate of initiation about twofold, although translational elongation was similar on all fiber messages. However, the rate of elongation of all fiber messages was threefold slower in abortively infected than in productively infected cells. This reduced elongation rate in abortive infections was specific for fiber. The similar distribution of fiber mRNAs on polysomes in both infections suggests that initiation must also be partially blocked in abortive infections. Since the majority of the fiber mRNA even in productive infections did not contain the ancillary leaders, the initiation and elongation defects in the abortive infection cannot be fully explained by the absence of these leaders. Therefore, other factors in the infected cell must be influencing the rate of translation.     

Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells.

Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection.     

Block to multiplication of adenovirus serotype 2 in monkey cells.

The block to adenovirus 2 (Ad2) multiplication in monkey cells can be overcome by coinfection with simian virus 40 (SV40). To identify this block we have compared the synthesis of Ad2 proteins in monkey cells infected with Ad2 alone (unenhanced) or with Ad2 plus SV40 (enhanced). Synthesis of viral proteins in enhanced cells was virtually identical to that found for permissive infection of human cells by Ad2 alone. In contrast, the unenhanced cells were strikingly deficient in the production of the IV (fiber) and 11.5K proteins whereas the synthesis of 100K and IVa2 was normal. Synthesis of a number of other proteins such as II, V, and P-VII was partially reduced. A similar specific reduction in synthesis of these proteins was found when their messages were assayed by cell-free translation. This result suggests that the block to Ad2 protein synthesis is at the RNA level rather than with the translational machinery of monkey cells. Analysis of the complexity and the concentration of Ak2-specific RNAs, using hybridization of restriction endonuclease fragments of the Ad2 genome to increasing concentrations of RNA, shows that although all species of late Ad2 mRNA are present, the concentration of several species is reduced sevenfold or more in unenhanced monkey cells as compared with enhanced cells. These species come from regions of the genome known to encode the deficient proteins. A model for the failure of adenovirus to multiply in monkey cells, based on abnormal processing of specific adenovirus messages, is presented.     

Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses.

The synthesis of simian virus 40 (SV40)-specific proteins in HeLa cells infected with the nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5, was investigated. Infected-cell proteins were labeled with radioactive amino acids late after infection, when host protein synthesis was shut off, and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All polypeptides normally seen in Ad2-infected cells were found in cells infected by the hybrid viruses. In addition to the Ad2-specific proteins, cells infected with Ad2+ND2 contain two SV40-specific proteins with apparent molecular weights of 42,000 and 56,000, cells infected with Ad2+ND4 contain one protein with an apparent molecular weight of 56,000, and cells infected with Ad2+ND5 contain one protein with an apparent molecular weight of 42,000. Cells infected with Ad2+ND3 do not contain detectable amounts of proteins not seen during Ad2 infection. Pulse-chase experiments demonstrate that the SV40-specific proteins induced by Ad2+ND2, Ad2+ND4, and Ad2+ND5 are metabolically unstable. These proteins are not present in purified virions. Two nonstructural Ad2-specific proteins have been demonstrated in Ad2 and hybrid virus-infected cells which have a smaller apparent molecular weight after a short pulse than after a pulse followed by a chase. The molecular weight increase during the chase may be caused by the addition of carbohydrate to a polypeptide backbone.     

Simian virus 40 (SV40)-specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U-antigen determinants.

The distribution of simian virus 40 (SV40)-specific proteins in nuclear subfractions of pulse-chase-labeled HeLa cells infected with nondefective adenovirus type 2 (Ad2)-SV40 hybrid viruses was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND5 specifically associate with the nuclear matrix and are virtually absent from the high-salt nuclear extract. In Ad2+ND4-infected HeLa cells, the SV40-specific proteins with molecular weights of 64,000 (64K) and lower also specifically associate with the nuclear matrix. The SV40-specific 72K, 74K, and 95K proteins were found both in the nuclear matrix and in the high-salt nuclear extract. Analyses of the nuclear matrices isolated from hybrid virus-infected cells by immunofluorescence microscopy showed that SV40 U-antigen-positive sera from SV40 tumor-bearing hamsters react with SV40-specific proteins integrated into nuclear matrices of HeLa cells infected by Ad2+ND1, Ad2+ND2, and Ad2+ND4, but not with nuclear matrices of HeLa cells infected by Ad2+ND5. This suggests that SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND4 integrated into the nuclear matrix carry SV40 U-antigen determinants. The apparent discrepancy in the subcellular localization of SV40-specific proteins in hybrid virus-infected cells when analyzed by biochemical cell fractionation procedures and when analyzed by immunofluorescence staining is discussed.     

Membrane interactions of simian virus 40 large T-antigen: influence of protein sequences and fatty acid acylation.

To sort out possible influences of protein sequences and fatty acid acylation on the plasma membrane association of simian virus 40 large T-antigen, we have analyzed the membrane interactions of carboxy-terminal fragments of large T-antigen, encoded by the adenovirus type 2 (Ad2+)-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. The 28,000 (28K)-molecular-weight protein of Ad2+ND1 as well as the 42K and 56K proteins of Ad2+ND2 associate preferentially with membranous structures and were found in association with the membrane system of the endoplasmic reticulum and with plasma membranes. Neither the endoplasmic reticulum membrane- nor the plasma membrane-associated 28K protein of Ad2+ND1 is fatty acid acylated. We, therefore, conclude that fatty acid acylation is not necessary for membrane association of this protein and suggest that an amino acid sequence in this protein is responsible for its membrane interaction. In contrast, the 42K and 56K proteins of Ad2+ND2 in plasma membrane fractions contain fatty acid. However, the interaction of these proteins with the plasma membrane differs from that of the 28K protein of Ad2+ND1: whereas the 28K protein of Ad2+ND1 interacts stably with Nonidet P-40-soluble constituents of the plasma membrane, the 42K and 56K proteins of Ad2+ND2 are tightly bound to the Nonidet P-40-insoluble plasma membrane lamina. Thus, an amino acid sequence in the amino-terminal region of the 28K protein confers membrane affinity to these proteins, whereas a region between the amino-terminal end of the 42K protein of Ad2+ND2 and the amino-terminal end of the 28K protein of Ad2+ND1 contains a reactive site for fatty acid acylation. This posttranslational modification correlates with the stable association of the 42K and 56K proteins with the plasma membrane lamina. We suggest that the same sequences also mediate the proper plasma membrane association of large T-antigen in simian virus 40-transformed cells.     

Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells.

The proteins that interact with cytoplasmic and nuclear polyadenylated RNA in adenovirus type 5 (Ad5) infection of HeLa cells were examined by UV-induced RNA-protein cross-linking in intact cells. The Ad5 100-kilodalton late nonvirion protein (100K protein) was cross-linked to both host and viral polyadenylated cytoplasmic RNA (mRNA). The cross-linking of the 100K protein to mRNA appears to correlate with productive infection, because the protein is not cross-linked to mRNA in abortive infection of wild-type Ad5 in monkey cells (CV-1) even though normal amounts of it are produced. However, when CV-1 cells are infected with Ad5 hr404, and Ad5 mutant which overcomes the host restriction to wild-type Ad5 infection in these cells, the 100K protein is cross-linked to mRNA. To identify and obtain antibodies to RNA-contacting proteins, a mouse was immunized with oligo(dT)-selected cross-linked RNA-protein complexes from Ad5-infected cells and the serum was used for immunoblotting experiments. It was found that in addition to the 100K protein, the Ad5 72K DNA-binding protein is also associated with RNA in the infected cells. The 72K DNA-binding protein is cross-linked to polyadenylated nuclear RNA sequences. These findings indicate that adenovirus proteins interact with RNAs in the infected cell and suggest possible mechanisms for the effects of the virus on mRNA metabolism.     

Biosynthesis, Immunological Specificity, and Intracellular Distribution of the Simian Virus 40-Specific Protein Induced by the Nondefective Hybrid Ad2+ND1

Ad2(+)ND(1), a nondefective hybrid virus containing a segment of the early region of simian virus 40 (SV40) DNA covalently inserted into the human adenovirus 2 genome, enhances the growth of human adenoviruses in simian cells and induces the SV40 U antigen. This hybrid previously has been shown to code for a 28,000 (28K) molecular weight protein not present in wild-type adenovirus 2-infected cells. By radioimmunoprecipitation using sera from hamsters bearing SV40-specific tumors, we have established that the Ad2(+)ND(1)-induced 28K protein is SV40-specific. This Ad2(+)ND(1)-induced protein is synthesized as a 30K molecular weight precursor, which is detectable only when infected cells are pulse-labeled in the presence of the protease inhibitor tosylamino phenylethyl chloromethyl ketone. Upon fractionation of labeled cell extracts, about 80% of the 28K protein is found in the plasma membrane fraction, whereas the remaining 20% is associated with the outer nuclear membrane. This protein is not detectable either in the nucleus or in the cytoplasm. Blockage of proteolytic cleavage by tosylamino phenylethyl chloromethyl ketone did not alter the topographic distribution of this SV40-specific protein, although the amount of the precursor protein in the outer nuclear membrane increased fourfold while that in the plasma membrane was proportionately decreased. This result suggests that the 28K protein is transferred from the outer nuclear membrane to the plasma membrane after posttranslational cleavage of the 30K precursor polypeptide. These data offer further support to the proposal that the 28K protein contains the determinants for SV40 U antigen and is responsible for SV40 enhancement of adenovirus growth in simian cells.     

Simian virus 40-specific polypeptides in AD2+ ND1- and Ad2+ ND4-infected cells.

A comparison of the proteins synthesized in human cells at late times after infection with adenovirus (Ad2) and with the adeno-simian virus 40 (SV40) hybrid viruses revealed polypeptides of 30,000 and 92,000 molecular weight specific for the hybrid viruses Ad2+ND1 and Ad2+ND4, respectively. Cell-free translation of SV40-specific mRNA, prepared from these cells by hybridization of total cytoplasmic RNA to SV40 DNA, showed that the mRNA's specifying these two polypeptides were at least partially encoded by the SV40 portion of the hybrid viruses. Cell-free translation of SV40-specific mRNA prepared from monkey cells infected with SV40 produced polypeptides of 40,000, 43,000, 48,500, and 92,000 molecular weight. The SV40 and Ad2+ND4 92,000-molecular-weight polypeptides made in vitro were very similar in electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to the polypeptide precipitated by Tegtmeyer (1974) with SV40 anti-T serum.     

Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2(+)ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2(+)ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2(+)ND1 either by the cleavage patterns produced by Escherichia coli R.RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2(+)ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2(+)ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2(+)ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40.     

Simian virus 40-specific ribosome-binding proteins induced by a nondefective adenovirus 2-simian virus 40 hybrid.

We have studied the intracellular distribution of the two simian virus 40-specific proteins, with apparent molecular weights of 56,000 and 42,000, detectable in human KB cells infected by a nondefective adenovirus 2-simian virus 40 hybrid, Ad2+ND2. After a 20-min pulse of [35S]methionine, about two-thirds of the newly synthesized 56K protein and one-third of the 42K protein were found localized on the plasma membrane. The remainder of each protein was found in the cytoplasm, whereas the nuclear fraction was virtually free of either component. A significant portion of both proteins present in the cytoplasmic fraction was complexed to the 40S ribosomal subunits and was not removed by treatment with 0.5 M KCl. Moreover, the portion that was found free in the cytoplasm could bind preferentially and quantitatively to purified 40S ribosomes in vitro, leading us to propose that these simian virus 40 proteins may act as translational control elements in cells.     

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.     

Genomic arrangement of an adenovirus-simian virus 40 hybrid virus, Ad2+ND4del.

Ad2+ND4del is an adenovirus type 2-simian virus 40 hybrid virus nondefective for growth in human cells. The virus was first observed when stocks of Ad2+ND4, a hybrid isolated from primary monkey kidney cells, were propagated in human cells. This paper describes the DNA sequence at two sites of DNA recombination, the site of the left adenovirus type 2-simian virus 40 junction and the site of a deletion of internal simian virus 40 sequences. Since the deletion was observed when the virus was switched from monkey to human cells, an analysis of gene expression in the region of DNA rearrangement may prove useful for the elucidation of molecular events that accompany virus growth in different hosts.     

Microinjection of mRNA enhances translational efficiency of human adenovirus fiber message in monkey cells.

In monkey cells abortively infected with human adenovirus serotype 2, the synthesis of the fiber polypeptide of the virion capsid is reduced by at least a factor of 100 when compared with that in monkey cells productively infected with a host range mutant of adenovirus serotype 2 (Ad2hr400). However, the steady-state level of fiber-encoding mRNA present in abortively infected monkey cells is only reduced by a factor of 5 to 10. When mRNA isolated from abortively and productively infected monkey cells was microinjected into the cytoplasms of uninfected or abortively infected monkey cells, no differences in the efficiency of translation of the fiber messages from these two sources were observed. These results suggest that the block to synthesis of the fiber polypeptide in abortively infected monkey cells does not reside in the translational machinery of the abortively infected cells themselves but may involve compartmentalization of the fiber message within the cells or an altered processing of the fiber message which prevents correct presentation to the ribosomes.