The Absence of Glycoprotein gL, but Not gC or gK, Severely Impairs Pseudorabies Virus Neuroinvasiveness

In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replicationcompetent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.For many alphaherpesviruses, four glycoproteins are required for virus entry into mammalian cells (32,52,53). In the case of herpes simplex virus (HSV) and bovine herpesvirus type 1, gB, gD, and the gH-gL heterodimer function independently or in concert to effect fusion of the virion envelope with the plasma membrane. All four glycoproteins are essential for the spread of these viruses from cell to cell and for cell fusion (2,35,46,57). However, little is known about the mechanism of these processes. Interestingly, although the same set of four glycoproteins are required for pseudorabies virus (PrV) entry, gD and gL are not essential for cell-cell spread (24, 25). Thus, HSV and PrV glycoproteins have evolved to have somewhat different functions.Klupp and Mettenleiter passaged a gL-null virus in cell culture and obtained a virus, PrV-⌬gLpass, that was able to enter cells, replicate, and spread from cell to cell (24). PrV-⌬gLpass had undergone gene rearrangements such that it lacked wildtype (WT) gH and instead contained a gDgH chimera resulting from a fusion between the first 271 amino acids of gD and the C-terminal 590 am...

Section: Resultsmentioning

confidence: 95%

Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras

Cairns

Milne

Ponce-de-León

et al. 2003

“…Clusters of infected and focally necrotic cells of the respiratory epithelium in the nasal cavity were detected at all time points investigated (24,48, and 56 h p.i.) (Fig.…”

Section: Kinetics Of Wild-type Prv Infectionmentioning

confidence: 99%

“…In contrast to the role of glycoproteins in herpesvirus neuroinvasion, which has been analyzed extensively in the past several years (1,2,3,4,24,34,63), no thorough examination of the role of viral tegument proteins in this process, except for US9 (11,12), has thus far been performed. Since gE is a major neurovirulence determinant in alphaherpesviruses and has also been implicated to play an important role in virion morphogenesis during secondary envelopment, it seems reasonable to assume that other proteins involved in virion maturation may also influence alphaherpesvirus neurotropism.…”

mentioning

confidence: 99%

Influence of Tegument Proteins of Pseudorabies Virus on Neuroinvasion and Transneuronal Spread in the Nervous System of Adult Mice after Intranasal Inoculation

Klopfleisch

Teifke

Fuchs

et al. 2004

Self Cite

Pseudorabies virus (PrV) is a neurotropic alphaherpesvirus that, after intranasal infection of adult mice, enters peripheral neurons and propagates to the central nervous system. In recent years we have analyzed the contribution of virus-encoded glycoproteins to neuroinvasion and transneuronal spread (reviewed in T. C. Mettenleiter, Virus Res. 92:197-206, 2003). We now extend our studies to analyze the role of tegument proteins in these processes. To this end, PrV mutants unable to express the UL11, UL37, UL46, UL47, and UL48 tegument proteins, as well as the corresponding rescued viruses, were intranasally instilled into 6-to 8-weekold CD1 strain mice. First, mean survival times were determined which showed that mice infected with the UL46 deletion mutant succumbed to the disease as early as wild-type PrV-infected animals. Survival times increased in the order: PrV-⌬UL47-, PrV-⌬UL11-, and PrV-⌬UL48-infected animals, a finding which parallels the growth phenotype of these viruses in cell culture. In contrast, none of the PrV-⌬UL37-infected animals died. Upon closer histological examination, all viruses except PrV-⌬UL37 were able to infect the nasal cavity and propagate to first-and second-order neurons as shown by two-color immunofluorescence. However, neuroinvasion was delayed in PrV-⌬UL47, PrV-⌬UL11, and PrV-⌬UL48, a finding that correlated with the extended survival times. Surprisingly, whereas PrV-⌬UL48 and PrV-⌬UL37 replicated to similar titers in cell culture which were ϳ500-fold lower than those of wild-type virus, after intranasal infection of mice PrV-⌬UL48 was able to infect areas of the brain like wild-type PrV, although only after a considerably longer time period. In contrast, PrV-⌬UL37 was not able to enter neurons and was restricted to the infection of single cells in the nasal respiratory epithelium. Thus, our data demonstrate the importance of herpesviral tegument proteins in neuronal infection and show a different contribution of tegument proteins to the neuroinvasion phenotype of a neurotropic alphaherpesvirus.Pseudorabies virus (PrV) is an alphaherpesvirus that causes Aujeszky's disease, a severe neurological disorder, in a broad range of mammalian species. The pig is the natural host of PrV since it is the only animal able to survive a productive infection, and latently infected pigs serve as a virus reservoir. After oronasal infection, which is the natural route of PrV entry into the organism, adult pigs usually show only mild clinical signs of respiratory distress or reproductive failure. In contrast, young piglets are very sensitive to virus invasion of the central nervous system (CNS) (36). Pruritus, the hallmark of the syndrome in nonporcine species, is usually not observed in pigs. In contrast to pigs, the course of infection in susceptible nonporcine species is peracute with a high mortality. These species are endstage hosts, i.e., they generally do not shed the virus and are therefore less important in transmission to other species (25,29,30,45,56,60).Since PrV infects a wide range o...

“…1B) (5). gK is required for virion release (18,42) but not for neuroinvasiveness (24), and gK Ϫ viruses are able to grow, albeit with reduced kinetics and yields compared with WT. It is thus possible that the slow growth phenotype of vJS⌬54 may result from reduced levels of gK expression.…”

mentioning

confidence: 99%

“…This does not account for the attenuation of vJS⌬54 in a mouse model of infection (Fig. 7), as gC is not required for penetration or propagation of PRV in the mouse nervous system (24).…”

mentioning

confidence: 99%

UL54 -Null Pseudorabies Virus Is Attenuated in Mice but Productively Infects Cells in Culture

Schwartz

Brittle

Reynolds

et al. 2006

The pseudorabies virus (PRV) UL54 homologs are important multifunctional proteins with roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. Here we describe the first genetic characterization of UL54. We constructed UL54 null mutations in a PRV bacterial artificial chromosome using sugar suicide and Red allele exchange systems. Surprisingly, UL54 is dispensable for growth in tissue culture but exhibits a small-plaque phenotype that can be complemented in trans by both the herpes simplex virus type 1 ICP27 and varicella-zoster virus open reading frame 4 proteins. Deletion of UL54 in the virus vJS⌬54 had no effect on the ability of the virus to shut off host cell protein synthesis but did affect virus gene expression. The glycoprotein gC accumulated to lower levels in cells infected with vJS⌬54 compared to those infected with wild-type virus, while gK levels were undetectable. Other late gene products, gB, gE, and Us9, accumulated to higher levels than those seen in cells infected with wild-type virus in a multiplicity-dependent manner. DNA replication is also reduced in cells infected with vJS⌬54. UL54 appears to regulate UL53 and UL52 at the transcriptional level as their respective RNAs are decreased in cells infected with vJS⌬54. Interestingly, vJS⌬54 is highly attenuated in a mouse model of PRV infection. Animals infected with vJS⌬54 survive twice as long as animals infected with wild-type virus, and this results in delayed accumulation of virus-specific antigens in skin, dorsal root ganglia, and spinal cord tissues.