Live Visualization of Herpes Simplex Virus Type 1 Compartment Dynamics

Abstract

We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.The herpes simplex virus type 1 (HSV-1) virion consists of three different compartments, capsid, tegument, and envelope. The icosahedral capsid has a diameter of 125 nm and contains the virus genome, a double-stranded DNA of 152 kbp. The structural basis of the capsid are the 162 capsomers, which include 150 hexons and 12 pentons (47). The capsomers are connected in groups of three by a complex formed with two copies of VP23 and one copy of VP19c (47,54,68). The hexons are composed of six molecules of the major capsid protein VP5. Eleven of the 12 pentons are composed of five molecules of VP5, while 1 of the 12, the so-called portal, is a cylindrical structure of 12 molecules of UL6 (46). Also involved in capsid assembly, but not physical components of the capsids, are the scaffold polypeptides VP22a, VP21, and the serine protease, VP24, which is required for capsid maturation (9,26,38,51). Six copies of VP26, a 12-kDa polypeptide encoded by the UL35 gene, occupy the tips of each hexon and thus decorate the surface of the capsid (42, 69). Although not essential...

A Functional Role for TorsinA in Herpes Simplex Virus 1 Nuclear Egress

“…The properties of wild-type HSV-1 strain F [HSV-1(F)] and HSV-1(F) bacterial artificial chromosome (BAC) genomes carrying a UL34 deletion (UL34-null) were described previously (46,56). A BAC expressing a monomeric red fluorescent protein (mRFP) fused to VP26 (rHSVBAC35R) was kindly provided by Cornel Fraefel (University of Zurich, Switzerland) (11). The VP5-null virus was a generous gift of Prashant Desai (12) and was propagated on VP5-expressing Vero cells.…”

Bovine herpesvirus 5 BICP0 complements the bovine herpesvirus 1 homolog

“…The Red recombination strategy with galK selection and counterselection permits efficient introduction of mutations into BACs (Warming et al, 2005) and has also been used before in our laboratory (de Oliveira et al, 2008). During the work described here, we experienced unexpected difficulties with this strategy when the entire BICP0-5 gene was used for insertion into the f115-d0-galK BAC.…”

“…To remove the bacterial sequences of the BAC, 0.05 µg of a Cre recombinase expressing plasmid (p116) was cotransfected (de Oliveira et al, 2008). One day after transfection of the BAC DNA, cells were examined in a Zeiss Axiovert S 100 fluorescence microscope.…”

“…In this BAC, the pBeloBAC11 sequence required for propagation in bacteria, adjacent to the eGFP gene under control of the hCMV IE promoter, has been inserted with flanking loxP sites between the BoHV-1 circ and BICP27 loci (Tobler et al, 2002). The protocol for Red recombination in E. coli strain SW102 was applied according to published procedures (de Oliveira et al, 2008;Warming et al, 2005). Briefly, in a first step, a DNA fragment containing the galK expression cassette flanked by 50-bp homology arms targeting the BoHV-5 specific frameshift (Fig.…”

“…To investigate the effect of the BoHV-5 specific frameshift mutation in the context of the BoHV-1 genome, we used the Red recombination strategy with galK selection and counterselection (de Oliveira et al, 2008). In the first step, the PCR-amplified galK cassette was inserted by homologous Red recombination into the parent f115(DF) BAC in place of a single guanosine at position 1924 in the BICP0-1 ORF (Fig.…”