Differing roles of inner tegument proteins pUL36 and pUL37 during entry of herpes simplex virus type 1

Abstract

Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17(+) did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.

FIG. 1.

Single-step virus growth. Replicate 35-mm dishes of complementing RS cells were infected with 10 PFU/cell of HSV-1 strain 17⁺ (WT), K5ΔZ, FRΔUL37, KΔUL36, or ARΔUL36 mutant virus. After 1 h at 37°C, the cells were washed at low pH to remove residual input infectivity and overlaid with 2 ml of DMEM, and incubation was continued at 37°C. Wild-type HSV-1 was grown on RS cells in an identical fashion as a control. At 3, 6, 12, and 24 h after infection, the cells were harvested by scraping into the supernatant medium, and the progeny virus was titrated on complementing cells.

FIG. 2.

Cytoplasmic capsids in infected cells. Replicate monolayers of HFFF₂ cells were infected with 5 PFU/cell of HSV-1 strain 17⁺ (WT) or K5ΔZ, FRΔUL37, KΔUL36, or ARΔUL36 mutant virus. Cells were fixed and prepared for electron microscopy at 24 h postinfection. Both free capsids (black arrowheads) and enveloped virions (white arrowheads) were present in the cytoplasm of WT HSV-1-infected cells. In addition, large numbers of virions were present on the cell surfaces (not shown). KΔUL36 and FRΔUL37 cytoplasmic capsids accumulated in aggregates, while ARΔUL36 capsids were dispersed individually throughout the cytoplasm. No enveloped virions were seen with any of the mutant viruses. Nuclear (nuc) and cytoplasmic (cyt) compartments are labeled. Bar = 1 μm.

FIG. 3.

KΔUL36 protein expression. (A) BHK cells were harvested 24 h after infection with 5 PFU/cell of HSV-1 strain 17⁺ (WT) or FRΔUL37, KΔUL36, or ARΔUL36 mutant virus. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with MAb E12-E3 directed against pUL36. The positions of full-length pUL36 and the 43-kDa N-terminal fragment (*) are indicated to the left of the gel and in the gel, and the positions of the protein size standards (in kilodaltons) are shown to the right of the gel. (B) Cytoplasmic capsids from KΔUL36-infected cells were separated by sucrose gradient sedimentation (see Fig. S2 in the supplemental material). The gradients were collected from the bottom in 30 equal fractions. Gradient fractions 3 to 27 were resolved by SDS-PAGE and analyzed by Western blotting. Blots were probed sequentially with MAb E12-E3 (anti-pUL36), DM165 (anti-VP5; to show the distribution of all capsid types), and MCA406 (anti-VP22a scaffolding protein; to show the location of B-capsids). The positions at which A-, B-, and C-capsids migrated are indicated below the blots. The positions of gradient fractions 5, 10, 15, 20, and 25 are shown above the blots.

FIG. 4.

Spread of virus infection. Replicate monolayers of HFFF₂ cells were infected with 0.01 PFU/cell of HSV-1 strain 17⁺ (WT) or K5ΔZ, FRΔUL37, KΔUL36, or ARΔUL36 mutant virus. (A) Unfused cells; (B) cells after treatment with PEG and dimethyl sulfoxide at 1 h postinfection to induce syncytium formation. The cells were fixed and labeled at 24 h postinfection. Viral DNA was visualized by FISH using Cy3-labeled probe (red), nuclei were stained with DAPI (blue), and cell cytoplasm was stained with CellMask deep red (yellow). Bars, 50 μm in all panels.

FIG. 5.

Capsid distribution in infected syncytia. Replicate monolayers of HFFF₂ cells were infected and induced to form syncytia as described in the legend to Fig. 4. Viral DNA was visualized by FISH using Cy3-labeled probe (red), and nuclei were stained with DAPI (blue). Bars, 50 μm in all panels.

FIG. 6.

Effect of nocodazole on spread of virus infection within syncytia. Replicate monolayers of HFFF₂ cells were infected with HSV-1 strain 17⁺ (WT) or FRΔUL37 mutant virus and induced to form syncytia as described in the legend to Fig. 4. Nocodazole (0.5 μg/ml) was added at 3 h postinfection, and incubation was continued for a further 21 h. For immunofluorescence, cells were fixed (54), and microtubule proteins were identified with MAb DM1A and Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (green). Viral DNA was visualized by FISH using Cy3-labeled probe (red). Nuclei were stained with DAPI (blue) The top panels show that the fibrillar microtubule network seen in uninfected syncytia [no nocodazole (−noc)] is disrupted by the addition of nocodazole (+noc). The bottom panels show the distribution of viral DNA in nocodazole-treated, WT HSV-1- and FRΔUL37-infected syncytia. Bars, 50 μm in all panels.

FIG. 7.

Protein content of virions and L-particles. Extracellular virions and L-particles from cells infected with HSV-1 strain 17⁺ (WT) and L-particles from cells infected with mutant virus K5ΔZ, FRΔUL37, KΔUL36, or ARΔUL36 were separated by Ficoll gradient sedimentation (see Fig. S4 in the supplemental material). The gradients were collected from the bottom in 10 equal fractions. A control sample of HSV-1 strain 17⁺ L-particles (LP) was run on each gel. Gradient fractions were resolved by SDS-PAGE and analyzed by Western blotting. Blots were probed sequentially with E12-E3 (anti-pUL36) (B), M780 (anti-pUL37) (C), and AGV031 (anti-pUL49) (A). The position of the 43-kDa N-terminal fragment of pUL36 is indicated in panel B.