To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic
Cre recombinase, as well as
beta-galactosidase expression cassettes. When the BAC pHSV1(17(+))blueLox was transfected back into eukaryotic cells, the
Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17(+))blueLox. We then tagged the
capsid protein VP26 and the envelope
protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17(+))blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged
proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral
glycoproteins, indicating that they were naked, cytosolic capsids. As the
infection progressed, they were enveloped and colocalized with the
viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope
proteins, and nuclear pores during a synchronous
infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.