Characterization of pseudorabies virus (PrV) cleavage-encapsidation proteins and functional complementation of PrV pUL32 by the homologous protein of herpes simplex virus type 1.

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.

Effects of simultaneous deletion of pUL11 and glycoprotein M on virion maturation of herpes simplex virus type 1.

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-DeltaUL11/gM-infected RK13 cells. The defects of HSV-1DeltaUL11 and HSV-1DeltaUL11/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.

Partial functional complementation of a pseudorabies virus UL25 deletion mutant by herpes simplex virus type 1 pUL25 indicates overlapping functions of alphaherpesvirus pUL25 proteins.

Homologs of the UL25 gene product of herpes simplex virus 1 (HSV-1) are highly conserved among the Herpesviridae. However, their exact function during viral replication is unknown. Current evidence suggests that in the alphaherpesvirus pseudorabies virus (PrV) the capsid-associated pUL25 plays a role in primary envelopment of DNA-containing mature capsids at the inner nuclear membrane. In the absence of pUL25, capsids were found in close association with the inner nuclear membrane, but nuclear egress was not observed (B. G. Klupp, H. Granzow, G. M. Keil, and T. C. Mettenleiter, J. Virol. 80:6235-6246, 2006). In contrast, HSV-1 pUL25 has been assigned a role in stable packaging of viral genomes (N. Stow, J. Virol. 75:10755-10765, 2001). Despite these apparently divergent functions, we wanted to assess whether the high sequence homology translates into functional homology. Therefore, we first analyzed a newly constructed HSV-1 UL25 deletion mutant in our assay system and observed a similar phenotype as in PrV. In the nuclei of infected cells, numerous electron-dense C capsids were detected, whereas primary envelopment of these capsids did not ensue. In agreement with results from PrV, vesicles were observed in the perinuclear space. Since these data indicated functional homology, we analyzed the ability of pUL25 of HSV-1 to complement a PrV UL25 deletion mutant and vice versa. Whereas a HSV-1 pUL25-expressing cell line partially complemented the pUL25 defect in PrV, reciprocal complementation of a HSV-1 UL25 deletion mutant by PrV pUL25 was not observed. Thus, our data demonstrate overlapping, although not identical functions of these two conserved herpesvirus proteins, and point to a conserved functional role in herpes virion formation.

Analysis of pseudorabies and herpes simplex virus recombinants simultaneously lacking the pUL17 and pUL25 components of the C-capsid specific component.

Homologs of the UL17 and UL25 gene products of herpes simplex virus 1 (HSV-1) are conserved throughout the Herpesviridae and essential for virus replication. However, their exact function is still unknown. Although both proteins form a complex on DNA-containing C-capsids defects observed in the absence of either protein differ. Absence of pUL17 from HSV-1 or the related alphaherpesvirus pseudorabies virus (PrV) precludes cleavage and packaging of newly replicated viral DNA, whereas in the absence of pUL25 genomic DNA is encapsidated but nuclear egress of capsids to the cytosol is abolished. HSV-1 pUL25 partially complemented the defect in a PrV UL25 deletion mutant indicating overlapping functions. However, reciprocal complementation did not ensue, and the present study demonstrates that UL17-deleted HSV-1 or PrV mutants are also not rescued by heterologous pUL17. To analyze whether simultaneous substitution of both complex partners may allow or increase trans-complementation we generated rabbit kidney cell lines co-expressing either PrV or HSV-1 pUL17 and pUL25, and respective HSV-1 and PrV double deletion mutants. Whereas the defects of both double mutants were trans-complemented by cell lines co-expressing the homologous complex partners, productive replication was not restored by heterologous pUL17 and pUL25. Thus, the protein complexes of PrV and HSV-1 either possess distinct functions, or require interactions with other viral proteins which are impaired in a heterologous context.

Phenotypic similarities and differences between UL37-deleted pseudorabies virus and herpes simplex virus type 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-DeltaUL37-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-1DeltaUL37[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-1DeltaUL37[86-1035] or PrV-DeltaUL37. Nuclear retention of nucleocapsids was not observed in either virus. However, in contrast to PrV-DeltaUL37, HSV-1DeltaUL37[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.

A capsid-modified helper-dependent adenovirus vector containing the beta-globin locus control region displays a nonrandom integration pattern and allows stable, erythroid-specific gene expression.

Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the beta-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated virus (AAV) inverted terminal repeats (Ad.LCR-beta-GFP). This vector possesses the adenovirus type 35 fiber knob that allows efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth factor-dependent CD34(+) erythroleukemic cell line, and in cord blood-derived human CD34(+) cells. Stable transduction of MO7e cells with Ad.LCR-beta-GFP was more efficient and less subject to position effects and silencing than transduction with a vector that did not contain the beta-globin LCR. Analysis of integration sites indicated that Ad.LCR-beta-GFP integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed integration sites were within the chromosomal beta-globin LCR. None of the Ad.LCR-beta-GFP integrations occurred in exons. The integration pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the beta-globin LCR-containing vector. Infection of primary CD34(+) cells with Ad.LCR-beta-GFP did not affect the clonogenic capacity of CD34(+) cells. Transduction of CD34(+) cells with Ad.LCR-beta-GFP resulted in vector integration and erythroid lineage-specific GFP expression.