The Herpes Simplex Virus Type 1 Glycoprotein D (gD) Cytoplasmic Terminus and Full-Length gE Are Not Essential and Do Not Function in a Redundant Manner for Cytoplasmic Virion Envelopment and Egress

Abstract

Herpes simplex virus type 1 (HSV-1) acquires its final envelope by budding into cytoplasmic vesicles thought to be derived from trans-Golgi network membranes. This process is facilitated by interactions among the carboxyl termini of viral glycoproteins and tegument proteins. To directly investigate the relative importance of the carboxyl terminus of glycoprotein D (gD) in the presence or absence of gE, a recombinant virus (gD⌬ct) was constructed to specify a truncated gD lacking the carboxy-terminal 29 amino acids. Furthermore, two additional recombinant viruses were constructed by mutating from ATG to CTG the initiation codons of gE (gEctg) or both gE and gM (gEctg؉gMctg), causing lack of expression of gE or both gE and gM, respectively. A fourth mutant virus was constructed to specify the gEctg؉gD⌬ct mutations. The replication properties of these viruses were compared to those of a newly constructed recombinant virus unable to express UL20 due to alteration of the two initiation codons of UL20 (UL20ctgctg). All recombinant viruses were constructed by using the double-Red, site-directed mutagenesis system implemented on the HSV-1(F) genome cloned into a bacterial artificial chromosome. The gEctg, gEctg؉gMctg, gD⌬ct, and gEctg؉gD⌬ct viruses produced viral plaques on African monkey kidney cells (Vero), as well as other cells, that were on average approximately 30 to 50% smaller than those produced by the wild-type virus HSV-1(F). In contrast, the UL20ctgctg virus produced very small plaques containing three to five cells, as reported previously for the ⌬UL20 virus lacking the entire UL20 gene. Viral replication kinetics of intracellular and extracellular viruses revealed that all recombinant viruses produced viral titers similar to those produced by the wild-type HSV-1(F) virus intracellularly and extracellularly at late times postinfection, with the exception of the UL20ctgctg and ⌬UL20 viruses, which replicated more than two-and-a-half logs less efficiently than HSV-1(F). Electron microscopy confirmed that all viruses, regardless of their different gene mutations, efficiently produced enveloped virions within infected cells, with the exception of the UL20ctgctg and ⌬UL20 viruses, which accumulated high levels of unenveloped virions in the cytoplasm. These results show that the carboxyl terminus of gD and the full-length gE, either alone or in a redundant manner, are not essential in cytoplasmic virion envelopment and egress from infected cells. Similarly, gM and gE do not function alone or in a redundant manner in cytoplasmic envelopment and virion egress, confirming previous findings.Herpes simplex virus type 1 (HSV-1) virion assembly initiates in the nucleus, where procapsid proteins assemble around scaffolding proteins (52, 60), followed by the digestion of these scaffolding proteins, creating empty capsids to be filled with DNA (31, 57, 58). The most-prevalent model for HSV-1 virion egress and viral envelope acquisition calls for primary envelopment of nuclear capsids as they cross the inner nuclear membran...

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K Is Required for Virion Entry via the Paired Immunoglobulin-Like Type-2 Receptor Alpha

“…Viral titers at different times postinfection were obtained on the VK302 cell line that expresses the HSV-1(KOS) gK gene. The ultrastructural morphology of virions within infected cells was examined by transmission electron microscopy as described previously (30,32,(34)(35)(36). All infected cells were processed at 18 h postinfection (hpi) and visualized by transmission electron microscopy.…”

“…Viral plaques were visualized by immunohistochemistry as we have previously described (29)(30)(31)(32). Analysis of one-step growth kinetics was performed as described earlier (20,22,33).…”

Phenylalanine Residues at the Carboxyl Terminus of the Herpes Simplex Virus 1 UL20 Membrane Protein Regulate Cytoplasmic Virion Envelopment and Infectious Virus Production

“…Using PCR test primers designed to lie outside the target mutation site(s), all mutated DNA regions were sequenced to verify the presence of the desired mutations in BACs. Similarly, viruses recovered from cells transfected with BACs (31) were sequenced to confirm the presence of the desired mutations. The entire genomes of the parental wild-type virus and the UL20F210A mutant viruses were sequenced using the Ion Torrent next-generation sequencing equipment (Life Technologies-Invitrogen; Carlsbad, CA), as we described previously (33).…”

“…Mutagenesis was accomplished in Escherichia coli cells using the markerless two-step Red recombination mutagenesis system and synthetic oligonucleotides (31,32) implemented on the bacterial artificial chromosome (BAC) plasmid pYEbac102-VC1 carrying the HSV-1(F) genome with gK and UL20 proteins tagged with V5 and 3ϫ FLAG antigenic epitopes, respectively. Unless otherwise specified, the wild-type virus used in all experiments is the VC-1 strain.…”

Analysis of contributions of herpes simplex virus type 1 UL43 protein to induction of cell-cell fusion

“…Using these primers to amplify the AphA1 gene located on pEPkan-S, a PCR product was produced that contained the desired mutation and the kanamycin resistance gene. Mutagenesis was accomplished by using a markerless two-step Red recombination mutagenesis system [11] implemented on the HSV-1 (F) bacterial artificial chromosome (BAC) as described previously [12,13]. The desired HSV-1 ΔUL43 was isolated from non-fluorescent progeny virus plaques.…”