Genital re-excretion of Murid gammaherpesvirus 4 following intranasal infection

in Proceedings of the 1st Scientific Meeting of the Faculty of Veterinary Medicine (2011, December 09)

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model ... [more ▼]

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4) in inbred laboratory mouse strains which are commonly accepted as a good model for studying gammaherpesviruses in vivo. To date, it has however never been possible to monitor viral reexcretion and virus transmission in this species. In order to identify potential re-excretion sites, intranasally infected mice were followed through global luciferase imaging for up to six months after infection. Surprisingly, we detected transient viral replication in mice genital tract at various times after latency establishment. Ex vivo imaging, quantitative PCR and immunohistochemistry revealed that virus genomes were present in high quantity in the vaginal tissue and that viral replication occurred mainly at the vaginal external border. Moreover, we highlighted the presence of free infectious viruses in the vaginal cavity at the moment of the observation of viral replication. As this ephemeral viral reexcretion could reveal a link with reproductive cycle, we compared reexcretion in normal and ovariectomized mice. Interestingly, no viral reactivation was observed in absence of hormonal cycle. In conclusion, we experimentally indentified for the first time a reexcretion site for MuHV-4 in mice that had been intranasaly infected. In the future, these results could help us to better understand the biology of gammaherpesviruses but should also allow us to develop strategies that could prevent the spread of these viruses in natural populations. [less ▲]

Bovine Herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects.

in Journal of Virology (2011), 85(2), 1011-1024

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's Sarcoma associated herpesvirus (KSHV) K8.1. In this study, we characterized that of ... [more ▼]

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's Sarcoma associated herpesvirus (KSHV) K8.1. In this study, we characterized that of Bovine Herpesvirus-4 (BoHV-4), encoded by the Bo10 gene. We identified a 180 kDa gene product, gp180, which was incorporated into the virion envelope. A Bo10 deletion virus was viable, but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since the Bo10 mutant was both less infectious for GAG(+) cells than the wild-type and more infectious for GAG(-) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the Murid Herpesvirus 4 gp150, and also to the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor. [less ▲]

A mechanistic basis for potent, glycoprotein B-directed gammaherpesvirus neutralization.

in Journal of General Virology (The) (2011), 92(Pt 9), 2020-33

Glycoprotein B (gB) is a conserved, essential component of gammaherpes virions and so potentially vulnerable to neutralization. However, few good gB-specific neutralizing antibodies have been identified ... [more ▼]

Glycoprotein B (gB) is a conserved, essential component of gammaherpes virions and so potentially vulnerable to neutralization. However, few good gB-specific neutralizing antibodies have been identified. Here, we show that murid herpesvirus 4 is strongly neutralized by mAbs that recognize an epitope close to one of the gB fusion loops. Antibody binding did not stop gB interacting with its cellular ligands or initiating its fusion-associated conformation change, but did stop gB resolving stably to its post-fusion form, and so blocked membrane fusion to leave virions stranded in late endosomes. The conservation of gB makes this mechanism a possible general route to gammaherpesvirus neutralization. [less ▲]

Comparative study of Murid gammaherpesvirus 4 infection in mice and in its natural host, the bank voles.

Poster (2009, December 11)

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. They are host-range specific and establish persistent, productive infections ... [more ▼]

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. They are host-range specific and establish persistent, productive infections of immunocompetent hosts. Thus, infected individuals simultaneously both elicit antiviral protective immune response and secrete infectious virions. The best studied gammaherpesviruses are Human herpesvirus 4 and Human herpesvirus 8. As these viruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4), a virus that has originally been isolated from bank voles (Myodes glareolus). Although MuHV-4 has not been isolated from house mice (Mus musculus), infection of inbred laboratory mouse strains is commonly accepted as a good model for studying gammaherpesviruses in vivo. It has however never been possible to monitor viral reexcretion and virus transmission in this species suggesting that this model could be imperfect. In this study, we therefore characterized MuHV-4 infection in its natural host, the bank voles, through classical virological methods but also through global luciferase imaging for an anatomical complete view of the infection. Results obtained show that, after intra-nasal infection, the natural route of infection is similar in mice and voles. Following nasal productive infection, the virus spreads to the lung where the infection is accompanied by massive cellular infiltrates. By opposition to extensive viral replication observed in mice, the different analyses indicated that the viral replication was ~1000 fold lower in bank voles. This lower replication did however not affect colonization of latency sites in superficial cervical lymph nodes and spleen as measured by real-time PCR quantification of viral genomes in these organs. In conclusion, this study revealed that MuHV-4 can experimentally infect bank voles, the supposed natural host, but with a lower replicative power. As, gammaherpesvirus epidemiology indicates that transmission correlates with the latent load, our results suggest that gammaherpesviruses may have evolved to infect their hosts without extensive lytic spread. In the future, establishment of experimental transmission in a population of Myodes glareolus should help us to better understand mechanisms used by gammaherpesviruses to evade immune response. [less ▲]

Glycoprotein L sets the neutralization profile of murid herpesvirus 4.

in Journal of General Virology (The) (2009), 90(Pt 5), 1202-14

Antibodies readily neutralize acute, epidemic viruses, but are less effective against more indolent pathogens such as herpesviruses. Murid herpesvirus 4 (MuHV-4) provides an accessible model for tracking ... [more ▼]

Antibodies readily neutralize acute, epidemic viruses, but are less effective against more indolent pathogens such as herpesviruses. Murid herpesvirus 4 (MuHV-4) provides an accessible model for tracking the fate of antibody-exposed gammaherpesvirus virions. Glycoprotein L (gL) plays a central role in MuHV-4 entry: it allows gH to bind heparan sulfate and regulates fusion-associated conformation changes in gH and gB. However, gL is non-essential: heparan sulfate binding can also occur via gp70, and the gB-gH complex alone seems to be sufficient for membrane fusion. Here, we investigated how gL affects the susceptibility of MuHV-4 to neutralization. Immune sera neutralized gL(-) virions more readily than gL(+) virions, chiefly because heparan sulfate binding now depended on gp70 and was therefore easier to block. However, there were also post-binding effects. First, the downstream, gL-independent conformation of gH became a neutralization target; gL normally prevents this by holding gH in an antigenically distinct heterodimer until after endocytosis. Second, gL(-) virions were more vulnerable to gB-directed neutralization. This covered multiple epitopes and thus seemed to reflect a general opening up of the gH-gB entry complex, which gL again normally restricts to late endosomes. gL therefore limits MuHV-4 neutralization by providing redundancy in cell binding and by keeping key elements of the virion fusion machinery hidden until after endocytosis. [less ▲]

Glycoprotein B switches conformation during murid herpesvirus 4 entry.

in Journal of General Virology (The) (2008), 89(Pt 6), 1352-63

Herpesviruses are ancient pathogens that infect all vertebrates. The most conserved component of their entry machinery is glycoprotein B (gB), yet how gB functions is unclear. A striking feature of the ... [more ▼]

Herpesviruses are ancient pathogens that infect all vertebrates. The most conserved component of their entry machinery is glycoprotein B (gB), yet how gB functions is unclear. A striking feature of the murid herpesvirus 4 (MuHV-4) gB is its resistance to neutralization. Here, we show by direct visualization of infected cells that the MuHV-4 gB changes its conformation between extracellular virions and those in late endosomes, where capsids are released. Specifically, epitopes on its N-terminal cell-binding domain become inaccessible, whilst non-N-terminal epitopes are revealed, consistent with structural changes reported for the vesicular stomatitis virus glycoprotein G. Inhibitors of endosomal acidification blocked the gB conformation switch. They also blocked capsid release and the establishment of infection, implying that the gB switch is a key step in entry. Neutralizing antibodies could only partially inhibit the switch. Their need to engage a less vulnerable, upstream form of gB, because its fusion form is revealed only in endosomes, helps to explain why gB-directed MuHV-4 neutralization is so difficult. [less ▲]

The murid herpesvirus-4 gH/gL binds to glycosaminoglycans.

in PLoS ONE (2008), 3(2), 1669

The first contact a virus makes with cells is an important determinant of its tropism. Murid Herpesvirus-4 (MuHV-4) is highly dependent on glycosaminoglycans (GAGs) for cell binding. Its first contact is ... [more ▼]

The first contact a virus makes with cells is an important determinant of its tropism. Murid Herpesvirus-4 (MuHV-4) is highly dependent on glycosaminoglycans (GAGs) for cell binding. Its first contact is therefore likely to involve a GAG-binding virion glycoprotein. We have previously identified two such proteins, gp70 and gp150. Gp70 binds strongly to GAGs. However, deleting it makes little difference to MuHV-4 cell binding or GAG-dependence. Deleting gp150, by contrast, frees MuHV-4 from GAG dependence. This implies that GAGs normally displace gp150 to allow GAG-independent cell binding. But the gp150 GAG interaction is weak, and so would seem unlikely to make an effective first contact. Since neither gp70 nor gp150 matches the expected profile of a first contact glycoprotein, our understanding of MuHV-4 GAG interactions must be incomplete. Here we relate the seemingly disconnected gp70 and gp150 GAG interactions by showing that the MuHV-4 gH/gL also binds to GAGs. gH/gL-blocking and gp70-blocking antibodies individually had little effect on cell binding, but together were strongly inhibitory. Thus, there was redundancy in GAG binding between gp70 and gH/gL. Gp150-deficient MuHV-4 largely resisted blocks to gp70 and gH/gL binding, consistent with its GAG independence. The failure of wild-type MuHV-4 to do the same argues that gp150 is normally engaged only down-stream of gp70 or gH/gL. MuHV-4 GAG dependence is consequently two-fold: gp70 or gH/gL binding provides virions with a vital first foothold, and gp150 is then engaged to reveal GAG-independent binding. [less ▲]

Glycosaminoglycan interactions in murine gammaherpesvirus-68 infection.

in PLoS ONE (2007), 2(4), 347

Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 ... [more ▼]

Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces. [less ▲]

Evidence for a multiprotein gamma-2 herpesvirus entry complex.

in Journal of Virology (2007), 81(23), 13082-91

Herpesviruses use multiple virion glycoproteins to enter cells. How these work together is not well understood: some may act separately or they may form a single complex. Murine gammaherpesvirus 68 (MHV ... [more ▼]

Herpesviruses use multiple virion glycoproteins to enter cells. How these work together is not well understood: some may act separately or they may form a single complex. Murine gammaherpesvirus 68 (MHV-68) gB, gH, gL, and gp150 all participate in entry. gB and gL are involved in binding, gB and gH are conserved fusion proteins, and gp150 inhibits cell binding until glycosaminoglycans are engaged. Here we show that a gH-specific antibody coprecipitates gB and thus that gH and gB are associated in the virion membrane. A gH/gL-specific antibody also coprecipitated gB, implying a tripartite complex of gL/gH/gB, although the gH/gB association did not require gL. The association was also independent of gp150, and gp150 was not demonstrably bound to gB or gH. However, gp150 incorporation into virions was partly gL dependent, suggesting that it too contributes to a single entry complex. gp150- and gL- gp150- mutants bound better than the wild type to B cells and readily colonized B cells in vivo. Thus, gp150 and gL appear to be epithelial cell-adapted accessories of a core gB/gH entry complex. The cell binding revealed by gp150 disruption did not require gL and therefore seemed most likely to involve gB. [less ▲]

Glycoprotein L disruption reveals two functional forms of the murine gammaherpesvirus 68 glycoprotein H.

in Journal of Virology (2007), 81(1), 280-91

The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and ... [more ▼]

The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. In order to define the role that gL plays in gamma-2 herpesvirus infections, we disrupted its coding sequence in murine gammaherpesvirus-68 (MHV-68). MHV-68 lacking gL folded gH into a conformation antigenically distinct from the form that normally predominates on infected cells. gL-deficient virions bound less well than the wild type to epithelial cells and fibroblasts. However, they still incorporated gH and remained infectious. The cell-to-cell spread of gL-deficient viruses was remarkably normal, as was infection, dissemination, and latency establishment in vivo. Viral membrane fusion was therefore gL independent. The major function of gL appeared to be allowing gH to participate in cell binding prior to membrane fusion. This function was most important for the entry of MHV-68 virions into fibroblasts and epithelial cells. [less ▲]