Effects of simultaneous deletion of pUL11 and glycoprotein M on virion maturation of herpes simplex virus type 1

Abstract

The conserved membrane-associated tegument protein pUL11 and envelope glycoprotein M (gM) are involved in secondary envelopment of herpesvirus nucleocapsids in the cytoplasm. Although deletion of either gene had only moderate effects on replication of the related alphaherpesviruses herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV) in cell culture, simultaneous deletion of both genes resulted in a severe impairment in virion morphogenesis of PrV coinciding with the formation of huge inclusions in the cytoplasm containing nucleocapsids embedded in tegument (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 78:3024-3034, 2004). To test whether a similar phenotype occurs in HSV-1, a gM and pUL11 double deletion mutant was generated based on a newly established bacterial artificial chromosome clone of HSV-1 strain KOS. Since gM-negative HSV-1 has not been thoroughly investigated ultrastructurally and different phenotypes have been ascribed to pUL11-negative HSV-1, single gene deletion mutants were also constructed and analyzed. On monkey kidney (Vero) cells, deletion of either pUL11 or gM resulted in ca.-fivefold-reduced titers and 40- to 50%-reduced plaque diameters compared to those of wild-type HSV-1 KOS, while on rabbit kidney (RK13) cells the defects were more pronounced, resulting in ca.-50-fold titer and 70% plaque size reduction for either mutant. Electron microscopy revealed that in the absence of either pUL11 or gM virion formation in the cytoplasm was inhibited, whereas nuclear stages were not visibly affected, which is in line with the phenotypes of corresponding PrV mutants. Simultaneous deletion of pUL11 and gM led to additive growth defects and, in RK13 cells, to the formation of large intracytoplasmic inclusions of capsids and tegument material, comparable to those in PrV-DeltaUL11/gM-infected RK13 cells. The defects of HSV-1DeltaUL11 and HSV-1DeltaUL11/gM could be partially corrected in trans by pUL11 of PrV. Thus, our data indicate that PrV and HSV-1 pUL11 and gM exhibit similar functions in cytoplasmic steps of virion assembly.

FIG. 1.

Construction of expression plasmids and virus mutants. (A) A schematic map of the HSV-1 genome shows the unique long (U_L) and unique short (U_S) regions flanked by terminal (TR_L and TR_S) and internal inverted (IR_L and IR_S) repeat sequences. Numbers denote kilobase pairs. Relevant ORFs (pointed rectangles) and restriction sites are indicated. (B) Eukaryotic expression plasmid pcDNA-HUL10 contains the gM gene flanked by the HCMV immediate-early promoter (P_HCMV) and a polyadenylation signal (pA). In pcDNA-HUL10K part of UL10 was replaced by a kanamycin resistance gene (KanR), and the PCR product obtained with P_T7- and P_SP6-specific primers was used for construction of gM deletion mutants. For preparation of a monospecific rabbit antiserum, the C terminus of gM was expressed as a bacterial fusion protein with GST from pGEX-HUL10. (C) Corresponding prokaryotic and eukaryotic expression plasmids were also generated for the complete UL11 gene (pGEX-HUL11 and pcDNA-HUL11). Deletion plasmid pcDNA-HUL11KF permitted removal of the KanR gene using flanking FRTs and introduction of a mutated start codon (CTG) as well as an artificial stop codon (TAG) to prevent expression of the 5′ part of UL11. (D) The genome of HSV-1 strain KOS was cloned as a BAC after insertion of a mini-F plasmid vector (pMBO131), together with an expression cassette for EGFP at the nonessential US5 gene encoding gJ. Artificial BamHI and PmeI restriction sites (shown in italic) were created to facilitate cloning and mutagenesis.

FIG. 2.

Western blot analyses. Purified virions of HSV-1 KOS or deletion mutants HSV-1ΔgM, HSV-1ΔUL11, and HSV-1ΔgM/UL11 were separated by SDS-polyacrylamide gel electrophoresis. After transfer to nitrocellulose filters, blots were probed with monospecific antisera against gM, pUL11, and pUL48. Locations of molecular mass markers are indicated on the left.

FIG. 3.

One-step growth analyses. Vero, Vero-UL11(HSV-1), RK13, and RK13-UL11(PrV) cells were infected with HSV-1 KOS, HSV-1ΔgM, HSV-1ΔUL11, and HSV-1ΔUL11/gM at an MOI of 5, harvested at the indicated times after infection, and titrated on Vero cells. Average titers and standard deviations from three independent experiments are shown.

FIG. 4.

Determination of plaque sizes. Plaque diameters of HSV-1 KOS, HSV-1ΔgM, HSV-1ΔUL11, and HSV-1ΔgM/UL11 on Vero, Vero-UL11 (HSV-1), RK13, and RK13-UL11 (PrV) cells were measured microscopically 3 days p.i. For each cell line, relative plaque sizes of all mutants were calculated by comparison to those of HSV-1 KOS, which were set as 100%. Average values and standard deviations from three independent experiments are shown.

FIG. 5.

Virion morphogenesis of HSV-1 KOS. RK13 (A to E) and Vero (F to H) cells were infected with HSV-1 KOS at an MOI of 1 and fixed and processed for electron microscopy 14 h after infection. Bars, 5 μm (A), 3 μm (F), 1 μm (H), 500 nm (B to E), and 300 nm (G).

FIG. 6.

Virion morphogenesis of HSV-1ΔgM. RK13 (A to C) and Vero cells (D and E) were infected at an MOI of 1 and fixed 24 h p.i. Bars, 3 μm (A and D), 500 nm (C), 300 nm (E), and 250 nm (B).

FIG. 7.

Virion morphogenesis of HSV-1ΔUL11. RK13 (A to C) and Vero cells (D to F) were infected at an MOI of 1 and fixed 24 h p.i. Bars, 3 μm (A and D), 1 μm (C and F), and 250 nm (B and E).

FIG. 8.

Virion morphogenesis of HSV-1ΔUL11/gM. RK13 (A and B) and Vero cells (C to E) were infected at an MOI of 1 and fixed 24 h p.i. Bars, 3 μm (A and C), 1 μm (E), 700 nm (B), and 200 nm (D).