The expression of N-terminal deletion DNA pilot proteins inhibits the early stages of phiX174 replication

Abstract

The phiX174 DNA pilot protein H contains four predicted C-terminal coiled-coil domains. The region of the gene encoding these structures was cloned, expressed in vivo, and found to strongly inhibit wild-type replication. DNA and protein synthesis was investigated in the absence of de novo H protein synthesis and in wild-type-infected cells expressing the inhibitory proteins (DeltaH). The expression of the DeltaH proteins interfered with early stages of DNA replication, which did not require de novo H protein synthesis, suggesting that the inhibitory proteins interfere with the wild-type H protein that enters the cell with the penetrating DNA. As transcription and protein synthesis are dependent on DNA replication in positive single-stranded DNA life cycles, viral protein synthesis was also reduced. However, unlike DNA synthesis, efficient viral protein synthesis required de novo H protein synthesis, a novel function for this protein. A single amino acid change in the C terminus of protein H was both necessary and sufficient to confer resistance to the inhibitory DeltaH proteins, restoring both DNA and protein synthesis to wild-type levels. DeltaH proteins derived from the resistant mutant did not inhibit wild-type or resistant mutant replication. The inhibitory effects of the DeltaH proteins were lessened by the coexpression of the internal scaffolding protein, which may suppress H-H protein interactions. While coexpression relieved the block in DNA biosynthesis, viral protein synthesis remained suppressed. These data indicate that protein H's role in DNA replication and stimulating viral protein synthesis can be uncoupled.

FIG. 1.

Schematic of the φX174 H protein. The predicted transmembrane and coiled-coil domains are depicted as gray and white boxes, respectively. The probability of each coiled-coil domain prediction (16) is given. The start codons of the N-terminal deletion proteins used in these studies are indicated by ΔH followed by the amino acid number found in the full-length protein. V286L represents the position and amino acid change of a mutation that confers resistance to the expression of the inhibitory ΔH proteins.

FIG. 2.

(A) Protein expression in wild-type-infected cells expressing the inhibitory ΔH142 protein. Lanes, from the left, contain purified virions (PV), whole-cell extracts of uninfected cells (UC), wild-type (WT)-infected cells with no exogenous protein expression (NP), wild-type-infected cells expressing the ΔH142 protein (ΔH), wild-type-infected cells expressing ΔH142 and internal scaffolding proteins (ΔHB), and wild-type-infected cells expressing the ΔH142V286L protein, which was derived from the resistant mutant (ΔH^R). (B) Protein expression in wild-type- and amber H mutant-infected cells. Lanes, from the left, contain whole-cell extracts of uninfected cells (UC), wild-type-infected cells (WT), am(H)Q26 mutant-infected cells, and am(H)E258 mutant-infected cells. The letter and number following am(H) indicate the codon in which the amber mutation is located. (C) Protein expression in wild-type- and φX174ΔH^RV286L-infected cells expressing the inhibitory ΔH142 protein. Lanes, from the left, contain whole-cell extracts of uninfected cells (UC), wild-type-infected cells (NP), wild-type-infected cells expressing the ΔH142 protein (ΔH), φX174ΔH^RV286L (resistant)-infected cells (NP), φX174ΔH^RV286L-infected cells expressing the ΔH142 protein (ΔH), and purified virions (PV).

FIG. 3.

Relative recovery of plasmid and viral RF DNAs in cells expressing the inhibitory ΔH142 protein. Plasmid and viral RF DNAs were isolated from infected cells and digested with SspI as described in the text. Lanes: 1, wild-type (WT)-infected cells expressing (+) the ΔH142 protein (ΔH) with inducer added at the time of infection (0); 2, digested RF DNA; 3, digested plasmid DNA (P); 4, wild-type-infected cells without induction of the inhibitory gene (−); 5, wild-type-infected cells expressing the ΔH142 protein (+) with inducer added 20 min before infection (−20); 6, amber H-infected cells (amH); 7, wild-type-infected cells coexpressing the internal scaffolding and inhibitory ΔH142 proteins (ΔHB); 8, wild-type-infected cells without induction of the internal scaffolding and inhibitory ΔH142 genes; 9, wild-type-infected cells expressing the ΔH142V286L protein, which was derived from the resistant mutant (ΔH^R); 10, wild-type-infected cells without induction of the ΔH142V286L gene; 11, φX174ΔH^RV286L (RM, resistant mutant)-infected cells expressing the ΔH142 protein; 12, φX174ΔH^RV286L-infected cells without induction of the ΔH142 gene; 13, digested plasmid DNA; 14, digested RF DNA.