The varicella-zoster virus ORFS/L (ORF0) gene is required for efficient viral replication and contains an element involved in DNA cleavage

Abstract

The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U(L)) and unique short (U(S)), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U(L) and U(S). VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U(L). An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U(L), directly adjacent to the a-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5' region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5' terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U(L), we concluded that our results can provide an explanation for the almost exclusive orientation of the U(L) seen in packaged VZV DNA.

FIG. 1.

Overview of the VZV ORFS/L genomic region and the mutants generated. (A) Schematic representation of the VZV genome with a focus on the terminal region containing ORFS/L. Scale bars provide an accurate measure of the genome and the expanded region. (B) Overview of the mutants generated with mutations in the ORFS/L region. A cross indicates the deletion of the corresponding region. Black arrows indicate the loci of stop codon or HA tag insertion.

FIG. 2.

Growth properties of vS/L_cHA and size determination of ORFS/L. (A) Multistep growth kinetics of vS/L_cHA and vP-Oka. Shown are the means and standard errors (error bars) of the results of three independent experiments. (B) Plaque size measurements of vS/L_cHA and vP-Oka. Shown are relative average plaque areas of 60 individual plaques with standard deviations (error bars), with the sizes of vP-Oka plaques set at 100%. (C) Western blot analysis of cells infected with vP-Oka (lane 1) or vS/L_cHA (lane 2). The arrow indicates the full-length ORFS/L protein, which has the predicted molecular mass.

FIG. 3.

Localization of ORFS/L in infected and transfected cells. (A) Localization of ORFS/L (green) is shown, along with GM130 (red), a marker for the Golgi apparatus, and DAPI (blue), for single infected MeWo cells (upper panels) or in syncytia (middle panels). The lower panels show a focus on the nuclei within a syncytium that shows colocalization of ORFS/L protein with the nuclear envelope. Scale bars correspond to 30 μm (upper panels) and 100 μm (middle panels). (B) MeWo cells were transfected with expression plasmid pORFS/L-His and analyzed 48 h after transfection. ORFS/L (green) and GM130 (red) were detected with the indicated antibodies. The transfected cells are representative of the cell population. Scale bar corresponds to 20 μm.

FIG. 4.

Growth properties of viruses lacking the entire ORFS/L. (A) Plaque size measurements of vΔaa1-157 (clone 1 [c1] and c2), vP-Oka, and revertant vΔaa1-157-rev generated from clone 1. Recombinant viruses were reconstituted in MeWo cells, IIF was performed 7 days after transfection, and images were recorded. Twenty individual plaque areas were measured and are shown as averages (vP-Oka plaques were set at 100%) with standard deviations (error bars). *, Plaque areas induced by Δaa1-157 mutants were significantly reduced (P < 0.0001) compared to those of parental vP-Oka or the revertant virus by Student's t test. (B) Representative plaque images for vΔaa1-157 (clones 1 and 2), vP-Oka, and vΔaa1-157-rev. Scale bars correspond to 200 μm.

FIG. 5.

Growth properties of a panel of ORFS/L mutants. (A) Multistep growth kinetics of indicated viruses. Virus replication of vaa1stop, vaa34stop, and vΔaa34-157 was significantly reduced (P < 0.05) at the indicated time points (asterisks) compared to that of vP-Oka or the corresponding revertant virus as determined by Student's t test. (B) Plaque size measurements of indicated viruses. *, Plaque areas of vaa1stop, vaa3stop, vaa34stop, and vΔaa34-157 were significantly reduced (P < 0.001) compared to those of vP-Oka (set to 100%) or the corresponding revertant virus as determined by Student's t test. Shown are the mean areas and standard deviations (error bars) for 60 individual plaques per virus. (C) Western blot analysis of cells infected with vP-Oka, vS/L_cHA, vaa1stop-cHA, vaa3stop-cHA, or vaa34stop-cHA. Samples were separated by SDS-15% PAGE and analyzed by Western blotting with anti-HA antibodies.

FIG. 6.

Cleavage of the viral genome in cells infected with mutant virus. (A) Schematic representation of the terminal fragments, TR_S and U_L, generated after restriction enzyme digestion with BamHI. The probe used to detect the genomic termini by Southern blotting is indicated. The scale bar provides an accurate measure of the sizes of the uncleaved (2.8 kbp), the TR_S (1.9 kbp), and the TR_L (0.9 kbp) fragments expected after cleavage and of the probe. (B) Cleavage analysis of whole-cell DNA derived from cells infected with vP-Oka, vaa1stop, or vaa1stop-rev. The P-Oka BAC (pP-Oka) served as a standard for uncleaved termini. The Southern blot shown is representative of the results of three independent experiments. (C) Cleavage analysis of nuclear DNA derived from cells infected with vP-Oka, vaa1stop, vaa1stop-rev, vaa3stop, or vaa34stop. The P-Oka BAC (pP-Oka) served as a standard for uncleaved termini. The Southern blot shown is representative of two independent experiments. (D) Quantification of band intensities of three independent cleavage assays using whole-cell DNA. The ratio of uncleaved to cleaved TR_L DNA fragments is shown relative to the results for vP-Oka. *, The relative amount of uncleaved termini of vaa1stop was significantly increased (P < 0.05) compared to the results for vP-Oka and vaa1stop-rev as determined by Student's t test. (E) Quantification of band intensities of two independent cleavage assays using nuclear DNA. The ratio of uncleaved to cleaved TR_L DNA fragments is shown relative to the results for vP-Oka.