Nuclear egress and envelopment of herpes simplex virus capsids analyzed with dual-color fluorescence HSV1(17+).
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.
Nagel
Claus-Henning
CH
Institute of Virology, OE 5230, Hannover Medical School, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany.
Döhner
Katinka
K
Fathollahy
Mojgan
M
Strive
Tanja
T
Borst
Eva Maria
EM
Messerle
Martin
M
Sodeik
Beate
B
D000818:Animals; D015139:Blotting, Southern; D002213:Capsid / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000821:virology; D002522:Chlorocebus aethiops; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D018259:Herpesvirus 1, Human / Q000254:growth & development / Q000378:metabolism*; D021381:Protein Transport