Mutation of the herpes simplex virus 1 KOS UL45 gene reveals dose dependent effects on central nervous system growth.

The herpes simplex virus type 1 (HSV-1) UL45 gene encodes an 18 kDa virion envelope protein whose function remains unknown. Previous studies using a UL45 null mutant, UL45A, demonstrated that deletion of the UL45 gene altered plaque size in Vero and HeLa cells, but was not essential for replication in these cell types. The goal of this study was to determine if mutation of the UL45 gene influenced virus growth in the CNS. Two UL45 mutants, as well as a repaired revertant virus, were constructed and tested for their ability to cause encephalitis and replicate in the CNS. The UL45 mutants were not lethal when 1 x 10(3) pfu were injected intracerebrally into Balb/c mice. In contrast, at inocula greater than 1 x 10(3), the UL45 mutants were lethal. In vivo growth curves derived from mice inoculated intracerebrally with 1 x 10(3) pfu of virus revealed that the mutants grew poorly compared to wild type or revertant viruses. These results suggest that the 18 kDa UL45 gene product is required for efficient growth in the central nervous system at low doses. We propose that the UL45 gene may play an important role under the conditions of a naturally acquired infection.

Membrane targeting properties of a herpesvirus tegument protein-retrovirus Gag chimera.

The retroviral Gag protein is capable of directing the production and release of virus-like particles in the absence of all other viral components. Budding normally occurs after Gag is transported to the plasma membrane by its membrane-targeting and -binding (M) domain. In the Rous sarcoma virus (RSV) Gag protein, the M domain is contained within the first 86 amino acids. When M is deleted, membrane association and budding fail to occur. Budding is restored when M is replaced with foreign membrane-binding sequences, such as that of the Src oncoprotein. Moreover, the RSV M domain is capable of targeting heterologous proteins to the plasma membrane. Although the solution structure of the RSV M domain has been determined, the mechanism by which M specifically targets Gag to the plasma membrane rather than to one or more of the large number of internal membrane surfaces (e.g., the Golgi apparatus, endoplasmic reticulum, and nuclear, mitochondrial, or lysosomal membranes) is unknown. To further investigate the requirements for targeting proteins to discrete cellular locations, we have replaced the M domain of RSV with the product of the unique long region 11 (U(L)11) gene of herpes simplex virus type 1. This 96-amino-acid myristylated protein is thought to be involved in virion transport and envelopment at internal membrane sites. When the first 100 amino acids of RSV Gag (including the M domain) were replaced by the entire UL11 sequence, the chimeric protein localized at and budded into the Golgi apparatus rather than being targeted to the plasma membrane. Myristate was found to be required for this specific targeting, as were the first 49 amino acids of UL11, which contain an acidic cluster motif. In addition to shedding new light on UL11, these experiments demonstrate that RSV Gag can be directed to internal cellular membranes and suggest that regions outside of the M domain do not contain a dominant plasma membrane-targeting motif.

Infection and replication of herpes simplex virus type 1 in an organotypic epithelial culture system.

We have used the organotypic culture system as a model to study the initial infectious process and spread of herpes simplex virus type 1 (HSV-1) in fully stratified and differentiated human epithelial tissue. The growth kinetics of HSV-1 were determined in organotypic tissues of human epidermal or ectocervical origin. Concurrently, we followed the spread of HSV-1 by immunostaining thin sections of infected organotypic tissue. After HSV-1 was applied to the top cornified epithelial layer, virus penetrated to the basal layer of replicating epithelium and grew to high titers. The virus was limited in its spread in that not all cells within the tissue had demonstrable infection. A ribonucleotide reductase mutant, ICP6 delta, could infect and replicate in basal layers of the organotypic tissues. However, we found that spread was limited in, and to, the basal cell layer. Peak ICP6 delta titers were 100-fold less than in cultures infected with wild-type HSV-1. Studies of HSV mutants should allow us to further define the role of specific viral genes which are associated with infection and spread in a tissue culture system that mimics the initial portal of entry for certain HSV infections.

Evidence for an interaction of herpes simplex virus with chondroitin sulfate proteoglycans during infection.

In a previous study, a mouse L cell mutant was isolated which is 90% resistant to HSV-1 infection (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro. J. Virol. 67, 93-100, 1993). This cell line, termed gro2C, failed to express heparan sulfate (HS)-glycosaminoglycans on the cell surface, which normally act as initial receptors for HSV-1 attachment to cultured cells. In this report, we extended the characterization of gro2C cells to explore the possibility that cell-surface chondroitin sulfate (CS) facilitates virus attachment to gro2C cells in the absence of HS. We found that soluble CS types A, B, and C strongly interfere with adsorption of HSV-1 to the surface of gro2C cells in a dose-dependent manner, and CS type B (dermatan sulfate) inhibited adsorption to parental (control) L cells by up to 10%. Moreover, gro2C cell infection was hypersensitive to inhibition by HS in comparison to control L cell infection. In all cases, a decrease in adsorption resulted in a decrease in infection. By contrast, the highly-sulfated glycosaminoglycan analog dextran sulfate was a relatively poor inhibitor of gro2C cell infection, indicating that the inhibitory effects of CS were related to its carbohydrate structure and not solely to its strong negative charge. By using a mutant virus strain which does not express the heparin-binding glycoprotein gC, we show that gC was not required for infection of gro2C cells, and was not required for the inhibition by HS or CS. Thus, the characterization of gro2C cell infection has revealed that one or more components of the HSV-1 particle can interact with cell-surface CS as well as HS to mediate infection of susceptible cells.

Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B.

Previous studies have shown that the initial interaction of herpes simplex virus (HSV) with cells is binding to heparan sulphate and that HSV-1 glycoprotein C (gC) is principally responsible for this binding. Although gC-negative viral mutants are impaired for binding and entry, they retain significant infectivity. The purpose of the studies reported here was to explore the requirements for infectivity of gC-negative HSV-1 mutants. We found that absence or alteration of cell surface heparan sulphate significantly reduced the binding of gC-negative mutant virus and rendered cells resistant to infection, as shown previously for the wild-type virus. We isolated a recombinant double-mutated HSV strain that produces virions devoid of both of the known heparin-binding glycoproteins, gB and gC. The drastically impaired binding of these mutant virions to cells, relative to gC-negative and wild-type virions, indicates that gB mediates the binding of gC-negative virions to cells. Thus at least two HSV glycoproteins can independently mediate the binding of HSV to cell surface heparan sulphate to start the process of viral entry into cells.

The HSV-1 UL45 18 kDa gene product is a true late protein and a component of the virion.

Previously we constructed a null mutation in the HSV-1 UL45 gene, showed that the UL45 gene was not required for growth in Vero cells, and confirmed that it coded for an 18 kDa protein (R.J. Visalli and C.R. Brandt, Virology 185:419-423, 1991). In this study, we have continued our characterization of the UL45 gene and the 18 kDa protein. Analysis of UL45 RNA revealed that the gene was expressed late and was inhibited in the presence of phosphonoacetic acid (paa), indicating it is a gamma 2 class gene. Using a specific polyclonal antiserum, we found that the 18 kDa UL45 gene product was also expressed late and was inhibited in the presence of paa. The 18 kDa protein was present in purified virions and was substantially enriched in the envelope-tegument fraction of virions disrupted with NP-40 detergent. The 18 kDa protein is thus a structural protein of the virus and appears to be associated with the viral envelope. A 20 kDa protein that cross-reacted with a polyclonal HSV-1 UL45 antiserum was also detected in cells infected with HSV-2 strain 333.

The HSV-1 UL45 gene product is not required for growth in Vero cells.

We have constructed a HSV-1 UL45 null mutant (UL45 delta) by inserting a TK-lacZ cassette into a BclI site near the 5' end of the UL45 gene. A polyclonal antiserum produced to an Escherichia coli trpE:UL45 fusion protein was used to show that an 18-kDa polypeptide corresponding to the predicted UL45 gene product was produced in HSV-1 strain KOS-infected Vero cells but was not detected in UL45 delta-infected Vero cells. The absence of the 18-kDa protein had only a slight effect on viral growth in cell culture, indicating that the UL45 gene product is not essential for growth in Vero cells. However, the burst size of UL45 delta was smaller than HSV-1 KOS in Vero and HeLa cells. UL45 delta also had a smaller plaque size and an altered plaque morphology.

The herpes simplex virus ribonucleotide reductase is required for ocular virulence.

We used a herpes simplex virus (HSV) type 1 ribonucleotide reductase (RR) null mutant (ICP6 delta) to study the role of HSV-1 RR in ocular HSV infections. We found that ICP6 delta was unable to induce vascularization of the cornea or stromal keratitis following inoculation into the cornea of BALB/c mice, but was able to induce a transient mild blepharitis. The parental strain (HSV-1 KOS) and a revertant of ICP6 delta, ICP6 delta+3.1, both caused severe ocular disease, indicating that HSV-1 RR is required for ocular virulence in mice. ICP6 delta grew poorly in vitro (Vero and BALB/c 3T3 fibroblasts) and in vivo (eye, trigeminal ganglia and brain) compared to ICP6 delta+3.1 and HSV-1 KOS, suggesting that the avirulence of ICP6 delta is due to poor growth in the host. ICP6 delta also grew less well in primary human corneal fibroblasts, suggesting that RR may be required for virulence in humans. These results indicate that drugs inhibiting the function of RR might be effective in treating ocular HSV infections.

Herpes simplex virus stromal keratitis is not titer-dependent and does not correlate with neurovirulence.

We developed a murine model of ocular herpes simplex virus (HSV) disease which is particularly suited for testing stromal keratitis because most animals show some evidence of infection. Using this model, we characterized the ocular disease patterns caused by ten recent low-passage clinical isolates of HSV-1, as well as those caused by the established laboratory strains HSV-1 KOS and HSV-2 333. Viral strains were evaluated for their ability to cause stromal keratitis, blepharitis, vascularization of the cornea, and mortality. The model was not useful for scoring epithelial keratitis. The ocular disease caused by the recent isolates ranged from very mild disease to severe stromal keratitis. Some of the recent isolates caused disease as severe as the two laboratory strains. A comparison of the virulence characteristics expressed by various HSV strains indicated that the ability to cause stromal disease was correlated with vascularization of the cornea (correlation coefficient = 0.797, P less than 0.001) and was not correlated with the neurovirulence of the strains (correlation coefficient 0.045, P greater than 0.05). The severity of stromal keratitis was not dependent on the amount of inoculum over the range tested and a strain causing severe stromal keratitis caused severe ocular disease even when mixed with a nonstromal strain at ratios of 10:1, 100:1, and 1000:1.