Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Jan;76(1):364-78.
doi: 10.1128/jvi.76.1.364-378.2002.

The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions

Walter Fuchs  1 , Barbara G Klupp, Harald Granzow, Nikolaus Osterrieder, Thomas C Mettenleiter
Affiliations

The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions

Walter Fuchs et al. J Virol. 2002 Jan.

Abstract

A 2.6-kbp fragment of the pseudorabies virus (PrV) genome was sequenced and shown to contain the homologues of the highly conserved herpesvirus genes UL31 and UL32. By use of a monospecific antiserum, the UL31 gene product was identified as a nuclear protein with an apparent molecular mass of 29 kDa. For functional analysis, UL31 was deleted by mutagenesis in Escherichia coli of an infectious full-length clone of the PrV genome. The resulting virus mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV. Ultrastructural analyses demonstrated that capsid maturation and DNA packaging were not affected. However, neither budding at the inner nuclear membrane nor cytoplasmic or extracellular virus particles were observed. These replication defects were similar to those of a UL34 deletion mutant (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein. Yeast two-hybrid studies revealed that a UL31 fusion protein specifically interacts with plasmids of a PrV genome library expressing the N-terminal part of UL34. Vice versa, UL34 selected UL31-encoding plasmids from the library. Immunofluorescence studies and immune electron microscopy demonstrated that in cells infected with wild-type PrV, both proteins accumulate at the nuclear membrane, whereas in the absence of UL34 the UL31 protein is dispersed throughout the nucleus. Like the UL34 protein, the UL31 gene product is a component of enveloped virus particles within the perinuclear space and absent from mature virions. Our findings suggest that physical interaction between these two virus proteins might be a prerequisite for primary envelopment of PrV at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Structure of the PrV genome and cloning strategies. (A) A schematic map of the PrV genome shows the long (UL) and short (US) unique regions, the inverted repeat sequences (IRS, TRS), and the positions of BamHI restriction sites. (B) The sequenced genome region of PrV-Ka (nt 1 to 2600; GenBank accession no. AJ319028) is part of plasmid pUC-SalIC. Primers PUL31-F and PUL31-R contain artificial EcoRI sites and were used to amplify the UL31 ORF for cloning in expression vectors. A bacterial GST fusion protein expressed from pGEX-UL31 was used for rabbit immunization (αUL31 serum). Plasmid pIRES-UL31 was used to generate a cell line (RK-UL31) expressing UL31 together with the neomycin resistance gene (neoR) under the control of the HCMV immediate-early gene promoter (PHCMV-IE) from a bicistronic mRNA which contains an intron (IVS) and an internal ribosomal entry site (IRES) between the two genes, and a polyadenylation signal [poly(A)] at the end. The T7 promoter (PT7) of pcDNA-UL31 permitted in vitro transcription and translation (IVT). In plasmids pcDNA-ΔUL31a and pcDNA-ΔUL31b, a major portion of UL31 was replaced by a kanamycin resistance gene (kanR) inserted in either orientation (pcDNA-ΔUL31a and -b). The modified gene was amplified by PCR with primers PUL31-F and PUL31-R and used for RecE- and RecT-mediated mutagenesis of pPrV-K1 (see panel C) in E. coli. (C) The mini-F plasmid pMBO1374 containing a chloramphenicol resistance gene (camR) was inserted into the cloned gG gene of PrV-Ka in plasmid pU6.3. The plasmid obtained, pU-ΔgGMBO, was used for generation of an infectious full-length clone of the PrV genome (pPrV-K1). After mutagenesis of the UL31 gene (pPrV-ΔUL31a and -b; see panel B), the vector insertion was removed by EcoRI digestion and cotransfection of cells with plasmid pU6.3. Thus, the resulting virus mutants (PrV-ΔUL31a and -b) contain an intact gG gene. (D) Plasmids pLex-UL31 and pLex-UL34 were utilized for yeast two-hybrid screening of a PrV genome library (pB42-XXX). Interactions between the fusion proteins were detected by LacZ expression from p8op-lacZ (Clontech), which is induced by binding of LexA to the LexA operator elements preceding the Gal 1 minimal promoter (PGal 1), and subsequent activation of transcription by the B42 protein part. Only relevant restriction sites are included in the maps in panels B to D. ORFs are drawn as pointed rectangles, and a slash indicates that the DNA fragment is not plotted to scale.
FIG. 2.
FIG. 2.
Identification of the UL31 protein of PrV. RK13 cells were infected with either PrV-Ka, PrV-ΔUL31a, or PrV-ΔUL34B (29) at an MOI of 5 and incubated for 8 h or the indicated times at 37°C. Lysates of infected cells, of noninfected RK13 and RK-UL31 cells (105 cells/lane), and of purified wild-type virions (V; 3 μg of protein/lane) were separated on SDS-12% polyacrylamide gels. Western blots were analyzed with monospecific rabbit antisera against UL31, UL34, and UL49. The digitally scanned images shown in panel A indicate that expression of UL31 and UL34 is not interdependent, whereas panel B illustrates the expression kinetics and the absence of the UL31 and UL34, but not of the UL49 proteins, from mature virions
FIG. 3.
FIG. 3.
In vitro growth properties of UL31 deletion mutants. (A) RK13 and RK-UL31 cells were fixed 48 h after infection under plaque assay conditions with PrV-Ka or PrV-ΔUL31a. Plaques were visualized by indirect immunofluorescence with a gC-specific monoclonal antibody. (B) One-step growth kinetics of PrV-Ka and of PrV-ΔUL31a and -b were determined in RK13 and RK-UL31 cells. At the indicated times after infection at an MOI of 10, the total amount of infectious intracellular and released progeny virus was determined by plaque assays on RK-UL31 cells.
FIG. 4.
FIG. 4.
Electron microscopy of cells infected with PrV-ΔUL31a. Noncomplementing RK13 cells (A and B) and trans-complementing RK-UL31 cells (C through E) were fixed 14 h after infection at an MOI of 1 and embedded in glycid ether 100. Ultrathin sections were stained with uranyl acetate and lead salts. In the absence of UL31, nucleocapsids are retained in the nucleus (A and B), whereas virus maturation in the cytoplasm (C and D) and release (C and E) are restored in UL31-expressing cells. Bars, 2.5 μm (A and C), 1 μm (B), or 500 nm (D and E).
FIG. 5.
FIG. 5.
Subcellular localization of the UL31 and UL34 proteins of PrV. Infected RK13 cells were incubated for 24 h (PrV-Ka) or 48 h (PrV-ΔUL31a and PrV-ΔUL34B) and fixed with a 1:1 mixture of methanol and acetone. Binding of αUL31 (A) or αUL34 (B) sera was detected by Alexa 488-conjugated secondary antibodies. For comparison, gC was visualized by a monoclonal antibody and TRITC-conjugated anti-mouse immunoglobulins. Green, red, and merged fluorescence reactions were analyzed by confocal laser scanning microscopy. Bars, 25 μm.
FIG. 6.
FIG. 6.
Nuclear localization of the UL31 and UL34 proteins. PrV-infected RK13 cells were fixed after 24 h (PrV-Ka) or 48 h (PrV-ΔUL31a, PrV-ΔUL34B) with a 1:1 mixture of methanol and acetone. Indirect immunofluorescence reactions of UL31- (A) and UL34 (B)-specific antisera and Alexa 488-conjugated secondary antibodies were analyzed by confocal laser scanning microscopy after chromatin counterstaining with propidium iodide. Green, red, and merged fluorescence reactions are imaged separately. Bars, 25 μm.
FIG. 7.
FIG. 7.
Localization of the UL31 protein within primary enveloped virus particles. RK13 cells were infected with PrV-Ka (A through C), PrV-ΔUS3 (D), or PrV-ΔUL31a (E) at an MOI of 1, fixed after 14 h, and embedded in Lowicryl K4M. After subsequent incubation with anti-UL31 serum and gold-tagged secondary antibodies, ultrathin sections were counterstained with uranyl acetate and analyzed by electron microscopy. The UL31 protein of PrV was detected along the nuclear membrane (A) and in primary enveloped virus particles in the perinuclear space (B and D) but not in released virions (C). In cells infected with PrV-ΔUL31a (E), no specific labeling was found. Bars, 200 nm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adler, H., M. Messerle, and U. H. Koszinowski. 2001. Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences. J. Virol. 75:5692–5696. - PMC - PubMed
    1. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C. Seguin, P. S. Tuffnell, and B. G. Barrell. 1984. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310:207–211. - PubMed
    1. Ben Porat, T., J. M. Demarchi, and A. S. Kaplan. 1974. Characterization of defective interfering viral particles present in a population of pseudorabies virions. Virology 61:29–37. - PubMed
    1. Ben Porat, T., R. A. Veach, and S. Ihara. 1983. Localization of the regions of homology between the genomes of herpes simplex virus type 1 and pseudorabies virus. Virology 127:194–204. - PubMed
    1. Berthomme, H., S. J. Monahan, D. S. Parris, B. Jacquemont, and A. L. Epstein. 1995. Cloning, sequencing, and functional characterization of the two subunits of the pseudorabies virus DNA polymerase holoenzyme: evidence for specificity of interaction. J. Virol. 69:2811–2818. - PMC - PubMed

Publication types

Associated data

LinkOut - more resources