Herpes simplex virus type 1 UL51 protein is involved in maturation and egress of virus particles

Abstract

The UL51 gene of herpes simplex virus type 1 (HSV-1) encodes a phosphoprotein whose homologs are conserved throughout the herpes virus family. Recently, we reported that UL51 protein colocalizes with Golgi marker proteins in transfected cells and that targeting of UL51 protein to the Golgi apparatus depends on palmitoylation of its N-terminal cysteine at position 9 (N. Nozawa, T. Daikoku, T. Koshizuka, Y. Yamauchi, T. Yoshikawa, and Y. Nishiyama, J. Virol. 77:3204-3216, 2003). However, its role in the HSV replication cycle was unknown. Here, we generated UL51-null mutants (FDL51) in HSV-1 to uncover the function of UL51 protein. We show that the mutant plaques were much smaller in size and that maximal titers were reduced nearly 100-fold compared to wild-type virus. Electron microscopy indicated that the formation of nucleocapsids was not affected by the deletion of UL51 but that viral egress from the perinuclear space was severely compromised. In FDL51-infected cells, a large number of enveloped nucleocapsids were observed in the perinuclear space, but enveloped mature virions in the cytoplasm, as well as extracellular mature virions, were rarely detected. These defects were fully rescued by reinsertion of the UL51 gene. These results indicate that UL51 protein is involved in the maturation and egress of HSV-1 virus particles downstream of the initial envelopment step.

FIG. 1.

Construction of the HSV-1 UL51 deletion mutant. A schematic map of the HSV-1 genome showing the long (U_L) and short (U_S) unique regions and the internal repeat (IR) and terminal repeat (TR) sequences. (A) Coding regions of proteins in the genomic region relevant for this study are shown. Arrows indicate transcriptional orientations. A recombination fragment containing the kanamycin resistance gene flanked by FRT sites (circled arrows) and UL51 homology regions is substituted for a substantial portion of the UL51 coding region from nucleotide positions 108277 to 108880. (B) Recombination results in the deletion of UL51 by replacement with the kanamycin resistance gene (FDL51Kan). Location of primers used for PCR genotyping is shown (Fig. 2). (C) Using Flp recombinase, the kanamycin resistance gene was excised, leaving behind one FRT sequence (FDL51). (D) Plasmid pBS1007 contains a BglII-EcoRI fragment of the HSV-1 genome used as a Southern blot probe and for the generation of the standard curve for the quantitative real-time PCR assay (Fig. 2).

FIG. 2.

Genotyping of the UL51 deletion mutant. (A) Agarose gel electrophoresis of PCR products containing the UL51 gene region. BAC plasmid DNA or viral genomic DNA was isolated and analyzed by PCR. Numbers below the gel photos indicate the PCR primer pairs used (Fig. 1B and C). (B) Location of the AscI fragment and schematic diagram of the neighboring loci of UL51. Arrows indicate transcriptional orientations. Thick bar represents the region hybridizing with the probe. (C) Southern blot analysis of AscI-digested DNA isolated from mock-, FDL51-, FDL51R-, YK304-, and HSV-1(F)-infected cells using the EcoRI-HindIII fragment of pBS1007 as a probe (Fig. 1D).

FIG. 3.

Quantitation of gene expression at loci neighboring UL51. (A) Real-time RT-PCR amplification profile of UL50, UL51, and UL52 mRNA in FDL51-infected Vero cells. Data analysis was performed using ABI Prism 7700 sequence detection system software (PE Applied Biosystems), with C_T values determined by automated threshold analysis. The fluorescence intensity collected in real time for each sample was plotted against the number of PCR cycles. The black horizontal line represents the threshold setting, set at 10 standard deviations above the baseline. The C_T value is defined as the fractional cycle number in which the fluorescence generated within a reaction has crossed the threshold. This value indicates that a sufficient number of amplicons have accumulated to a statistically significant point above the baseline. C_T values correlate inversely with target gene expression. To assess relative differences in gene transcript levels, 18S rRNA was used as an RNA control. (B) Standard linear regression for HSV-1 genes. Serially diluted pBS1007 was amplified as described in Materials and Methods. Amplification was detected, and the C_T was analyzed by linear regression analysis. The formula and correlation coefficient used are shown in each panel. (C) Log copy numbers of mRNA from UL51-neighboring genes in HSV mutant-infected cells. Shown are average values from two separate experiments in which samples were run in duplicate. Transcript levels from real-time PCR were converted to mRNA copy number.

FIG. 4.

Expression of major tegument proteins. Vero cells were infected with FDL51, FDL51R, YK304, or HSV-1(F) at an MOI of 3. The cells were harvested at 24 h postinfection, and an equivalent amount of protein from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting with rabbit antiserum against UL51, UL48 (VP16), UL46, or UL49 (VP22) proteins. Molecular mass markers are shown to the left. The arrowheads indicate the position of each protein.

FIG. 5.

Growth properties of FDL51 in Vero cells. (a) Plaque formation of FDL51 mutant virus. Vero cells were infected with FDL51, FDL51R, YK304, or HSV-1(F) and were incubated in a medium containing gamma globulin for 48 h. Viral plaques were photographed at 48 h postinfection, and the mean diameters of 20 single plaques per virus mutant were determined as described in Materials and Methods. (b to e) Photos of viral plaques. Vero cells were infected with FDL51 (b), FDL51R (c), YK304 (d), and HSV-1(F) (e). Bars, 100 nm in all panels. (f and g) Growth kinetics of FDL51. Vero cells were infected with FDL51, FDL51R, YK304, or HSV-1(F) at an MOI of 0.01 (f) or 3 (g). At each time point, cells and supernatants were harvested and progeny virus titers were determined by plaque assays, as described in Materials and Methods. The average results of two independent experiments are shown.

FIG. 6.

TEM analysis of extracellular regions of FDL51-infected cells. Vero cells were infected at an MOI of 3 with FDL51 (a, b, and d) or HSV-1(F) (c and e) and analyzed by electron microscopy 24 h postinfection. In FDL51-infected cells, extracellular mature virions were rarely observed in cell-to-cell junctions (b) or on the surfaces of infected cells (a and d), in contrast to wild-type HSV-1(F)-infected cells (c and e). Bars, 2 μm (a to c) or 0.2 μm (d and e).

FIG. 7.

TEM analysis of FDL51-infected Vero cells. Vero cells were infected with FDL51 (a and c to f) or HSV-1(F) (b) and were analyzed by TEM as described in Materials and Methods. In FDL51-infected cells, nucleocapsids in the cytoplasm (a, arrows) and intracytoplasmic vesicles with enveloped matured virions (a, arrowheads) were rarely observed, while they were abundant in wild-type HSV-1(F)-infected cells (b, arrows and arrowheads). Insets show high-magnification views of the squares. No obvious difference was seen in the intranuclear nucleocapsids between cells infected with FDL51 (c and d, arrows) and those with wild-type virus (data not shown). Large numbers of enveloped nucleocapsids accumulated in the perinuclear region of FDL51-infected cells (c and d, thick arrows). A membrane-bound intranuclear structure containing enveloped nucleocapsids (e) and an interwoven membrane-like structure were occasionally observed near the margin of the nucleus in FDL51-infected cells (f). Arrows nucleocapsid; arrowheads, enveloped mature virion in the cytoplasm; thick arrows, enveloped nucleocapsid in the perinuclear region. Bars, 1 μm (a and b) or 0.5 μm (c to f).

FIG. 8.

Distribution of virus particles. Numbers of virus particles present in the different compartments of FDL51-infected Vero cells at 24 h postinfection were determined. Particles were counted in electron micrographs of 20 randomly selected cells for wild-type and FDL51-infected cells. Bars show the average particle numbers in each compartment. (a) Nucleocapsids in the nucleus; (b) enveloped nucleocapsids in the perinuclear space; (c) nucleocapsids in the cytoplasm; (d) enveloped mature virions in the cytoplasm; (e) extracellular mature virions on the outer surface of the plasma membrane.