Helena Browne

An analysis of the in vitro and in vivo phenotypes of mutants of herpes simplex virus type 1 lacking glycoproteins gG, gE, gI or the putative gJ.

Mutants of herpes simplex virus type 1 (HSV-1) lacking glycoproteins gG, gE, gI or the putative gJ were constructed by inserting a lacZ expression cassette within the US4, US8, US7 and US5 genes respectively. Revertant viruses were then constructed by rescue with a wild-type DNA fragment. Each of these mutant viruses, by comparison with the parental virus HSV-1 SC16, exhibited normal particle to infectivity ratios, and had no discernible phenotypic abnormalities in baby hamster kidney-21 cells following high or low multiplicity infections. Infection of mice by scarification of the ear with these mutant viruses showed the following. (i) Interruption of the US5 (gJ) gene has no effect on the ability of HSV-1 to multiply at the inoculation site or its ability to enter or multiply in the peripheral or central nervous system (CNS). This shows that the US5 gene provides a convenient site for the insertion of foreign genes for both in vitro and in vivo studies. (ii) Disruption of the US4 (gG) gene results in marginal attenuation in the mouse ear model. (iii) Disruption of the US7 (gI) or US8 (gE) genes results in pronounced attenuation; virus was rapidly cleared from the inoculation site and was barely detectable in sensory ganglia or in the CNS. The failure of gI-negative or gE-negative viruses to replicate efficiently at the inoculation site in vivo led to the investigation of virus behaviour in epithelial cells in vitro. Viruses lacking gE or gI adsorbed to and entered these cells at normal rates compared with the parental virus, but formed minute plaques. This is consistent with a failure of cell-to-cell spread by the cell contact route. This was confirmed by measurement of the rate of increase in infectious centre numbers following low multiplicity infections. The view that gE and gI influence interactions between cells at the plasma membrane was reinforced by showing that the introduction of disrupted gE or gI genes into a syncytial, but otherwise syngeneic, background resulted in a non-syncytial phenotype. We conclude that the gE-gI complex plays a part, at least in some cell types, in the interactions at the cell surface that allow transmission of the virus from infected to uninfected cells by cell contact. In syncytial strains this leads to uncontrolled membrane fusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Herpes Simplex Virus Nucleocapsids Mature to Progeny Virions by an Envelopment → Deenvelopment → Reenvelopment Pathway

ABSTRACT Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.

Localization of HCMV UL33 and US27 in Endocytic Compartments and Viral Membranes

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor‐like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor‐like proteins, UL33 and US27, in transfected and human cytomegalovirus‐infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody‐feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus‐infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus‐infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.

Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL.

Herpes simplex virus type 1 (HSV-1) glycoproteins gB, gD and gHL are capable of inducing cell fusion when expressed from plasmid vectors in the absence of any other virus components. Fusion requires the expression of all four glycoproteins on the same membrane, since they are unable to cooperate in trans to induce syncytium formation. In addition, the fusion event is dependent on the expression of a gD receptor on target cell membranes and does not require the presence of cell-surface glycosaminoglycans.

Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells

BackgroundHuman Cytomegalovirus (HCMV) has acquired through evolution a number of genes to try to evade immune recognition of the virus-infected cell. Many of these mechanisms act to inhibit the MHC class I antigen presentation pathway, but any virus-infected cell which has down-regulated cell surface expression of MHC class I proteins, to avoid CTL attack, would be expected to become susceptible to lysis by Natural Killer cells. Surprisingly, however, HCMV infected fibroblasts were found to be resistant to NK cell mediated cytotoxicity. Expression of the UL16 glycoprotein could represent one mechanism to help the virus to escape from NK cell attack, as it has been shown to bind, in vitro, some of the ligands for NKG2D, the NK cell activating receptor. Here, we explored the role of UL16, in the context of a viral infection, by comparing the susceptibility to NK lysis of cells infected with HCMV and cells infected with a UL16 deletion mutant of this virus.ResultsCells infected with the UL16 knockout virus were killed at substantially higher levels than cells infected with the wild-type virus. This increased killing could be correlated with a UL16-dependent reduction in surface expression of ligands for the NK cell activating receptor NKG2D.ConclusionsExpression of the UL16 glycoprotein was associated with protection of HCMV-infected cells from NK cell attack. This observation could be correlated with the downregulation of cell surface expression of NKG2D ligands. These data represent a first step towards understanding the mechanism(s) of action of the UL16 protein.

Herpes simplex virus tegument protein VP16 is a component of primary enveloped virions.

ABSTRACT Immunogold electron microscopy was used to determine whether the tegument proteins VP13/14, VP22, and VP16 of herpes simplex virus type 1 (HSV1) are components of primary enveloped virions. Whereas VP13/14 and VP22 were not detected in virus particles in the perinuclear space and were present in only mature extracellular virions, VP16 was acquired prior to primary envelopment of the virus at the inner nuclear membrane. This finding highlights potential similarities and differences between HSV1 and the related alphaherpesvirus, pseudorabies virus, in which the homologues of all three of these tegument proteins are not incorporated into the virion until secondary envelopment.

The Transmembrane Domain and Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein H Play a Role in Membrane Fusion

ABSTRACT Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.

Evidence for a role of the membrane-proximal region of herpes simplex virus type 1 glycoprotein h in membrane fusion and virus inhibition

We have identified a putative membrane‐interacting domain preceding the transmembrane domain of the Herpes simplex virus type 1 (HSV‐1) glycoprotein H (gH). Peptides derived from this region interact strongly with membranes and show a high tendency to partition at the interface. This region is predicted to bind at the membrane interface by adopting an α helical structure. Peptides representing either the HSV‐1 gH pretransmembrane region or a scrambled control with a different hydrophobic profile at the point of interface have been studied. The peptides derived from this domain of gH induce the fusion of liposomal membranes, adopt helical conformations in membrane mimetic environments and are able to inhibit HSV‐1 infectivity. The pretransmembrane region appears to be a common feature in viral fusion proteins of several virus families, and such a feature might be related to their fusogenic function. The identification of membrane‐interacting regions capable of modifying the biophysical properties of phospholipid membranes lends weight to the view that such domains might function directly in the fusion process and could facilitate the future development of HSV‐1 entry inhibitors.

Analysis of a Membrane Interacting Region of Herpes Simplex Virus Type 1 Glycoprotein H

Glycoprotein H (gH) of herpes simplex virus type I (HSV-1) is involved in the complex mechanism of membrane fusion of the viral envelope with the host cell. Membrane interacting regions and potential fusion peptides have been identified in HSV-1 gH as well as glycoprotein B (gB). Because of the complex fusion mechanism of HSV-1, which requires four viral glycoproteins, and because there are only structural data for gB and glycoprotein D, many questions regarding the mechanism by which HSV-1 fuses its envelope with the host cell membrane remain unresolved. Previous studies have shown that peptides derived from certain regions of gH have the potential to interact with membranes, and based on these findings we have generated a set of peptides containing mutations in one of these domains, gH-(626–644), to investigate further the functional role of this region. Using a combination of biochemical, spectroscopic, and nuclear magnetic resonance techniques, we showed that the α-helical nature of this stretch of amino acids in gH is important for membrane interaction and that the aromatic residues, tryptophan and tyrosine, are critical for induction of fusion.

Analysis of the L1 Gene Product of Human Papillomavirus Type 16 by Expression in a Vaccinia Virus Recombinant

The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.