Exploring the Nuclear Envelope of Herpes Simplex Virus 1-Infected Cells by High-Resolution Microscopy

Abstract

Herpesviruses are composed of capsid, tegument, and envelope. Capsids assemble in the nucleus and exit the nucleus by budding at the inner nuclear membrane, acquiring tegument and the envelope. This study focuses on the changes of the nuclear envelope during herpes simplex virus 1 (HSV-1) infection in HeLa and Vero cells by employing preparation techniques at ambient and low temperatures for high-resolution scanning and transmission electron microscopy and confocal laser scanning microscopy. Cryo-field emission scanning electron microscopy of freeze-fractured cells showed for the first time budding of capsids at the nuclear envelope at the third dimension with high activity at 10 h and low activity at 15 h of incubation. The mean number of pores was significantly lower, and the mean interpore distance and the mean interpore area were significantly larger than those for mock-infected cells 15 h after inoculation. Forty-five percent of nuclear pores in HSV-1-infected cells were dilated to more than 140 nm. Nuclear material containing capsids protrude through them into the cytoplasm. Examination of in situ preparations after dry fracturing revealed significant enlargements of the nuclear pore diameter and of the nuclear pore central channel in HSV-1-infected cells compared to mock-infected cells. The demonstration of nucleoporins by confocal microscopy also revealed fewer pores but focal enhancement of fluorescence signals in HSV-1-infected cells, whereas Western blots showed no loss of nucleoporins from cells. The data suggest that infection with HSV-1 alters the number, size, and architecture of nuclear pores without a loss of nucleoporins from altered nuclear pore complexes.

Epstein-Barr Virus Protein Kinase BGLF4 Targets the Nucleus through Interaction with Nucleoporins

“…Almost half of the nuclear pores in herpes simplex virus 1-infected cells were larger than 140 nm, with the protrusion of some nuclear material into the cytoplasm being detected. However, it remains unclear whether viral capsids with an average size of 115 to 130 nm can bud through the dilated pores or whether the virus egresses from the nucleus through an envelope-dependent process and the coupled NPC enlargement may facilitate the transport of other materials required for nucleocapsid assembly (60). In a preliminary experiment, the nuclear retention of yellow fluorescent protein (YFP)-LacZ-NLS was not affected by the expression of BGLF4 (data not shown).…”

BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex To Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins

“…Alterations in nuclear shape and size were found in HSV-1-infected cells (74). HSV-1 infection also induced morphological alterations in nuclear pores, visualized by electron microscopy, resulting in the loss of pore numbers and an increase in dilated gaps in the nuclear membrane (75). In HCMV-infected cells, a change in the shape of the nucleus and leakage of 155-kDa dextran into the nucleus were found at the nuclear concave regions, close to the cytoplasmic assembly compartment (76).…”

High-Pressure Freezing: Current State and Future Prospects

“…Freeze-fracturing of high-pressure frozen samples is usually performed by sandwich fracturing [ 26 ]. A random fracture face is produced by hitting off one carrier of the complete specimen carrier sandwich rather than creating fracture faces by using the knife in the freeze-fracturing machine ( see Note 9 ).…”