Nuclear Egress and Envelopment of Herpes Simplex Virus Capsids Analyzed with Dual-Color Fluorescence HSV1(17 ⁺ )

Abstract

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 ␤-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.Herpes simplex virus type 1 (HSV1) causes severe human diseases such as herpes encephalitis or herpes keratoconjunctivitis (18). Its double-stranded DNA genome of 152 kb that codes for more than 80 open reading frames is enclosed in an icosahedral capsid with a diameter of 125 nm. HSV1 is a spherical, enveloped virus with a diameter of about 250 nm. Between the capsid and the viral membrane, there is an amorphous, asymmetric layer, the tegument, which consists of about 20 different proteins (45,74). HSV1 enters cells by fusion at the plasma membrane or after endocytosis by fusion with an endosomal membrane (19,42,66,67,82). After dynein-mediated transport along microtubules (32,56,59,81), capsids reach the nuclear pore where the viral genome is released into the nucleoplasm (68) for viral transcription and DNA replication (74). Progeny viral genomes are packaged into preassembled nuclear capsids, which translocate to the inner nuclear membrane. The subsequent steps of herpesvirus morphogenesis are controversial (12, 13, 65).HSV1 capsids can leave the nucleus by primary envelopment at the inner nuclear membrane (6, 64). According to the luminal or single-envelopment hypothesis, these enveloped virions present in the lumen of the nuclear envelope or the endoplasmic reticulu...

Contribution of Direct and Cross-Presentation to CTL Immunity against Herpes Simplex Virus 1

“…Moreover, the entire cytoplasm was labeled by gDGFP, indicating the synthesis of HSV1 envelope proteins in the endoplasmic reticulum (Fig. 1D), and there was a strong perinuclear gD signal that most likely represented the trans-Golgi network where HSV1 capsids acquire their final envelope (27). However, we were unable to detect any infectious virus particle in the supernatant of HSV1-inoculated DC by plaque assay using Vero cells (data not shown), suggesting that the DC synthesized structural viral proteins, but did not release infectious HSV1.…”

“…Such dual-color viral strains allow the detection of all cells and subcellular structures containing viral proteins, irrespective of their interaction and possible masking by host factors or other structural viral proteins (27). Moreover, they do not require specific Abs directed against viral proteins, which often cross-react with cellular structures in primary cells.…”

“…Wild-type (wt) HSV1 strain F (ATCC VR-733; HSV1(F)-wt; cf (27) were amplified in BHK cells and titrated in Vero ϩ DC by HSV1 using reporter gene LacZ. Inoculation with increasing MOI per DC led to integration, transcription, and eventual translation of ␤-galactosidase, whose activity was measured using its substrate ONPG.…”

“…Escherichia coli DY380 harboring pHSV1(17 ϩ )Lox, a bacterial artificial chromosome (BAC) in which the thymidine locus of pHSV1(17ϩ)blueLox (27) had been repaired and the ␤-galactosidase expression cassette had been removed (N. Müther, C.-H. Nagel, B. Sodeik, and M. Messerle, unpublished data), were grown in the presence of 17 g/ml chloramphenicol at 32°C. For BAC mutagenesis, the red recombination system of bacteriophage , which has been inserted into the genome of E. coli DY380 (30), was induced by heat shock for 15 min at 42°C, then the bacteria were cooled on ice and made electrocompetent.…”

DNA-tumor virus entry—From plasma membrane to the nucleus

“…For example, the gamma-herpesvirus murine herpesvirus 4 which lacks the ORF75c tegument protein failed to be transported to the nucleus of murine cells although it was delivered into the cytosol (75). Incoming HSV1 capsids preferentially recruit the dynein/dynactin motor complex as opposed to a plus end-directed motor, whereas newly assembled capsids recruit a periphery directed motor (76,77).…”