scholarly journals Characterization of Pseudorabies Virus (PrV) Cleavage-Encapsidation Proteins and Functional Complementation of PrV pUL32 by the Homologous Protein of Herpes Simplex Virus Type 1

2009 ◽  
Vol 83 (8) ◽  
pp. 3930-3943 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Tobias Leege ◽  
Thomas C. Mettenleiter
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Pseudorabies Virus ◽  
Gene Products ◽  
Homologous Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Cleavage and encapsidation of newly replicated herpes simplex virus type 1 (HSV-1) DNA requires several essential viral gene products that are conserved in sequence within the Herpesviridae. However, conservation of function has not been analyzed in greater detail. For functional characterization of the UL6, UL15, UL28, UL32, and UL33 gene products of pseudorabies virus (PrV), the respective deletion mutants were generated by mutagenesis of the virus genome cloned as a bacterial artificial chromosome (BAC) in Escherichia coli and propagated in transgenic rabbit kidney cells lines expressing the deleted genes. Neither of the PrV mutants was able to produce plaques or infectious progeny in noncomplementing cells. DNA analyses revealed that the viral genomes were replicated but not cleaved into monomers. By electron microscopy, only scaffold-containing immature but not DNA-containing mature capsids were detected in the nuclei of noncomplementing cells infected with either of the mutants. Remarkably, primary envelopment of empty capsids at the nuclear membrane occasionally occurred, and enveloped tegument-containing light particles were formed in the cytoplasm and released into the extracellular space. Immunofluorescence analyses with monospecific antisera of cells transfected with the respective expression plasmids indicated that pUL6, pUL15, and pUL32 were able to enter the nucleus. In contrast, pUL28 and pUL33 were predominantly found in the cytoplasm. Only pUL6 could be unequivocally identified and localized in PrV-infected cells and in purified virions, whereas the low abundance or immunogenicity of the other proteins hampered similar studies. Yeast two-hybrid analyses revealed physical interactions between the PrV pUL15, pUL28, and pUL33 proteins, indicating that, as in HSV-1, a tripartite protein complex might catalyze cleavage and encapsidation of viral DNA. Whereas the pUL6 protein is supposed to form the portal for DNA entry into the capsid, the precise role of the UL32 gene product during this process remains to be elucidated. Interestingly, the defect of UL32-negative PrV could be completely corrected in trans by the homologous protein of HSV-1, demonstrating similar functions. However, trans-complementation of UL32-negative HSV-1 by the PrV protein was not observed.

scholarly journals Phenotypic similarities and differences between UL37-deleted pseudorabies virus and herpes simplex virus type 1

2009 ◽  
Vol 90 (7) ◽  
pp. 1560-1568 ◽  
Author(s):  
Tobias Leege ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Pseudorabies Virus ◽  
Wild Type Virus ◽  
Rabbit Kidney ◽  
Simplex Virus ◽  
Hsv 1

In the absence of the tegument protein pUL37, virion formation of pseudorabies virus (PrV) and herpes simplex virus type 1 (HSV-1) is severely impaired. Non-enveloped nucleocapsids accumulate in clusters in the cytoplasm, whereas only a few enveloped particles can be detected. Although a contribution of pUL37 to nuclear egress of HSV-1 has been suggested, the nuclear stages of morphogenesis are not impaired in PrV-ΔUL37-infected cells. Moreover, HSV-1 pUL37 has been described as essential for replication, whereas PrV is able to replicate productively without pUL37, although to lower titres than wild-type virus. Thus, there may be functional differences between the respective pUL37 proteins. This study compared the phenotypes of UL37-deleted PrV and HSV-1 in parallel assays, using a novel pUL37 deletion mutant of HSV-1 strain KOS, HSV-1ΔUL37[86–1035]. Aggregates of seemingly ‘naked’ nucleocapsids were present in the cytoplasm of African green monkey (Vero) or rabbit kidney (RK13) cells infected with HSV-1ΔUL37[86–1035] or PrV-ΔUL37. Nuclear retention of nucleocapsids was not observed in either virus. However, in contrast to PrV-ΔUL37, HSV-1ΔUL37[86–1035] was unable to replicate productively in, and to form plaques on, either Vero or RK13 cells. Trans-complementation of respective deletion mutants with the heterologous pUL37 did not ensue. These data demonstrate that the conserved pUL37 in HSV-1 and PrV have similar but distinct functions.

scholarly journals Characterization of heparan sulphate 3-O-sulphotransferase isoform 6 and its role in assisting the entry of herpes simplex virus type 1

2005 ◽  
Vol 385 (2) ◽  
pp. 451-459 ◽  
Author(s):  
Ding XU ◽  
Vaibhav TIWARI ◽  
Guoqing XIA ◽  
Christian CLEMENT ◽  
Deepak SHUKLA ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Cho Cells ◽  
Heparan Sulphate ◽  
Simplex Virus ◽  
Entry Receptor ◽  
Hsv 1

Heparan sulphate (HS) 3-O-sulphotransferase transfers sulphate to the 3-OH position of the glucosamine residue of HS to form 3-O-sulphated HS. The HS modified by 3-O-sulphotransferase isoform 3 binds to HSV-1 (herpes simplex virus type 1) gD (envelope glycoprotein D), and the resultant 3-O-sulphated HS serves as an entry receptor for HSV-1. In the present paper, we report the isolation and characterization of a novel HS 3-O-sulphotransferase isoform, designated HS 3-O-sulphotransferase isoform 6 (3-OST-6). Mouse 3-OST-6 gene was identified in the EST (expressed sequence tag) database and cloned into pcDNA3.1/Myc-His vector. A CHO (Chinese-hamster ovary) cell line that stably expresses 3-OST-6 (3OST6/CHO cells) was prepared. The disaccharide analysis of the HS isolated from 3OST6/CHO cells revealed that 3-OST-6 exhibits HS 3-O-sulphotransferase activity. Furthermore, 3OST6/CHO cells were susceptible to infection by HSV-1, but not by other alphaherpesviruses examined, suggesting that 3-OST-6 produces a specific entry receptor for HSV-1. Our results indicate that a new member of 3-OST family generates an entry receptor for HSV-1. The findings add to the growing body of evidence that HSV-1 entry is mediated by 3-O-sulphated HS generated by multiple members of 3-O-sulphotransferases.

scholarly journals Effects of Simultaneous Deletion of pUL11 and Glycoprotein M on Virion Maturation of Herpes Simplex Virus Type 1

2008 ◽  
Vol 83 (2) ◽  
pp. 896-907 ◽  
Author(s):  
Tobias Leege ◽  
Walter Fuchs ◽  
Harald Granzow ◽  
Martina Kopp ◽  
Barbara G. Klupp ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Pseudorabies Virus ◽  
Single Gene ◽  
Rabbit Kidney ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The conserved membrane-associated tegument protein pUL11 and envelope glycoprotein M (gM) are involved in secondary envelopment of herpesvirus nucleocapsids in the cytoplasm. Although deletion of either gene had only moderate effects on replication of the related alphaherpesviruses herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV) in cell culture, simultaneous deletion of both genes resulted in a severe impairment in virion morphogenesis of PrV coinciding with the formation of huge inclusions in the cytoplasm containing nucleocapsids embedded in tegument (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 78:3024-3034, 2004). To test whether a similar phenotype occurs in HSV-1, a gM and pUL11 double deletion mutant was generated based on a newly established bacterial artificial chromosome clone of HSV-1 strain KOS. Since gM-negative HSV-1 has not been thoroughly investigated ultrastructurally and different phenotypes have been ascribed to pUL11-negative HSV-1, single gene deletion mutants were also constructed and analyzed. On monkey kidney (Vero) cells, deletion of either pUL11 or gM resulted in ca.-fivefold-reduced titers and 40- to 50%-reduced plaque diameters compared to those of wild-type HSV-1 KOS, while on rabbit kidney (RK13) cells the defects were more pronounced, resulting in ca.-50-fold titer and 70% plaque size reduction for either mutant. Electron microscopy revealed that in the absence of either pUL11 or gM virion formation in the cytoplasm was inhibited, whereas nuclear stages were not visibly affected, which is in line with the phenotypes of corresponding PrV mutants. Simultaneous deletion of pUL11 and gM led to additive growth defects and, in RK13 cells, to the formation of large intracytoplasmic inclusions of capsids and tegument material, comparable to those in PrV-ΔUL11/gM-infected RK13 cells. The defects of HSV-1ΔUL11 and HSV-1ΔUL11/gM could be partially corrected in trans by pUL11 of PrV. Thus, our data indicate that PrV and HSV-1 pUL11 and gM exhibit similar functions in cytoplasmic steps of virion assembly.

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