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. 2009 Oct;83(20):10582-95.
doi: 10.1128/JVI.00497-09. Epub 2009 Aug 5.

Open reading frame 33 of a gammaherpesvirus encodes a tegument protein essential for virion morphogenesis and egress

Haitao Guo  1 , Lili Wang, Li Peng, Z Hong Zhou, Hongyu Deng
Affiliations

Open reading frame 33 of a gammaherpesvirus encodes a tegument protein essential for virion morphogenesis and egress

Haitao Guo et al. J Virol. 2009 Oct.

Abstract

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.

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Figures

FIG. 1.
FIG. 1.
Characterization of MHV-68 ORF33. (A) MHV-68 ORF33 is expressed as a true late gene. The upper panel shows a Northern blot analysis of ORF33 mRNA from MHV-68-infected BHK-21 cells using a probe for ORF33 coding region. Lanes 1 to 3, lane 6, and lane 8 show samples at 0, 8, 12, 24, and 36 h postinfection, respectively; lane 4, 12 h postinfection plus 100 μg of CHX/ml; lane 5, 12 h postinfection plus 200 μg of CHX/ml; lane 7, 24 h postinfection plus 200 μg of PAA/ml; and lane 9, 32 h postinfection plus 200 μg of PAA/ml. 28S rRNA was used as a loading control. The lower panel shows a Western blot analysis of the ORF33 protein from MHV-68-infected BHK-21 cells using anti-ORF33 polyclonal serum. Lane 1, mock infection; lanes 2 to 6 and lane 8: 0, 4, 8 12, 24, and 32 h postinfection, respectively; lane 7, 24 h postinfection plus PAA treatment; and lane 9, 32 h postinfection plus PAA. β-Actin was used as a loading control. (B) Expression of ORF33 in transfected cells. 293T cells were mock transfected or transfected with pFLAG-33 (the upper panel), pEGFP-C1 vector, or pEGFP-33 (the lower panel). Cells were collected at 48 h after transfection and subjected to Western blotting with anti-FLAG antibody or anti-GFP antibody. Actin was probed as a loading control. (C) Cellular localization of ORF33 in transfected cells. 293T cells were transfected with pFLAG-33 and then analyzed by indirect immunofluorescence assay (panel 1). BHK-21 cells were transfected with pEGFP-33 and then either mock infected (panel 2) or infected with MHV-68 at an MOI of 5 PFU/cell for 24 h (panels 3 and 4). Fluorescence was observed with a confocal laser microscope. (D) Effect of detergent treatment or trypsin digestion on the association of ORF33 with purified virions. The purified virions were treated with trypsin or detergent, as described in Materials and Methods. The tegument-nucleocapsid complexes were analyzed by Western blotting sequentially using anti-gB, anti-ORF45, anti-ORF33, and anti-ORF26 polyclonal antibodies.
FIG. 2.
FIG. 2.
Construction and analysis of ORF33-null MHV-68 (BAC) DNAs. (A) Nucleotide sequence of the region containing the mutation in the ORF33-null MHV-68 mutant. The genome coordinates are given to the left of the nucleotide sequence and on top of the nucleotides between which mutations were introduced. The ORF33 ATG and the three inserted nonsense and frameshift mutations are boxed, and the introduced VspI site is underlined. (B) Schematic representation of the ORF33 locus and its flanking ORFs on the WT BAC or 33STOP genome, the position of a probe (nt 49109 to 50264), and the predicted restriction fragments detected by the probe in Southern blotting analysis. (C) Southern blotting analysis of WT BAC and 33STOP DNAs. Various BAC DNAs were digested with VspI or BglII as indicated at the top of the panel, electrophoresed, blotted, and hybridized with the DIG-labeled probe. Positions of a λ/HindIII DNA ladder run on the same gel are indicated to the left of the panel.
FIG. 3.
FIG. 3.
Deficiency in and transcomplementation of viral replication for ORF33-null mutant. (A) 293T cells were transfected with WT BAC plus plasmid pEGFP-C1 (panels 1 and 1′), 33STOP plus pEGFP-C1 (panels 2 and 2′), 33STOP plus pEGFP-33 (panels 3 and 3′), or 33STOP.R plus pEGFP-C1 (panels 4 and 4′). Cell morphology and GFP fluorescence were visualized under a fluorescence microscope at 4 days posttransfection, as indicated to the left. (B) Deficiency in infectious virus production by 33STOP. Supernatants from 293T cells transfected with various BAC DNAs plus pEGFP-C1 or pGFP-33 were collected at 4 days posttransfection. Virus titers were determined by plaque assays in BHK-21 cells in duplicates. Error bars represent standard deviations from three independent experiments. (C) 293T cells were transfected with various BAC DNAs plus pEGFP-C1 or pGFP-33. At the indicated time points, total cellular DNA was isolated and analyzed by a real-time PCR assay using primers specific to ORF65 sequences. The copy number of viral genomes was normalized to 25 ng of total cellular DNA. Error bars represent the standard deviations from three independent experiments. (D) The deficiency of 33STOP in virion release can be rescued in trans. Extracellular virions DNAs and total cellular DNAs were separately isolated from 293T cultures transfected with various BAC DNAs plus pEGFP-C1 or pEGFP-33 at 5 days posttransfection. The copy number of viral genomes was determined by a real-time PCR assay using primers specific to ORF65. The copy number of cell associated viral genomes was normalized to 25 ng of total cellular DNA, and the copy number of extracellular viral genomes was expressed as viral genome copies per 1 ml of supernatant. Error bars represent the standard deviations from three independent experiments. (E) 293T cells were individually transfected with WT BAC plus plasmid pCMV-HA, 33STOP plus pCMV-HA, 33STOP plus pHA-33, or 33STOP.R plus pCMV-HA. At 4 days posttransfection, the cells were lysed and probed by Western blotting with polyclonal anti-MHV-68 serum, anti-ORF26, anti-ORF65, anti-ORF33, anti-ORF45, or anti-ORF52 antibodies or a monoclonal anti-HA antibody. Actin was probed as a loading control.
FIG. 4.
FIG. 4.
ORF33 is not required for MHV-68 lytic DNA replication. (A) Analysis of viral DNA replication in viral genome transfection experiment. 293T cells were transfected with WT BAC plus pCMV-HA, 33STOP plus pCMV-HA, 33STOP plus pHA-33 or 33STOP.R plus pCMV-HA. At the indicated time points posttransfection, total cellular DNA was isolated and analyzed by a real-time PCR assay using primers specific to ORF65. The copy number of viral genomes was normalized to 25 ng of total cellular DNA. (B) Determining virus titers in the supernatant from the transfection experiment. The supernatant from 293T cells transfected with WT BAC, 33STOP, or 33STOP.R plus pCMV-HA was collected at 24 or 36 h posttransfection. Virus titers were determined by plaque assay in BHK-21 cells. (C) Analysis of viral DNA replication in infection experiment. BHK-21 cells were infected with the WT BAC, 33STOP, or 33STOP.R virus at 100 genome copies per cell. At 12, 24, 36, or 48 h postinfection, total DNAs were isolated from the infected cells. The copy number of viral genomes was determined by real-time PCR. The result is presented as the fold increase in viral genome copy numbers at 24, 36, and 48 h postinfection compared to that at 12 h postinfection. (D) Determining the virus titer in the supernatant from the infection experiment. The supernatant from BHK-21 cells infected with WT BAC, 33STOP, or 33STOP.R virus was collected at 0, 12, 24, 36, or 48 h postinfection. Virus titers were determined by plaque assay in BHK-21 cells. No plaque was detected in supernatant from 33STOP infection at any of the time points. Error bars represent the standard deviations from three independent experiments.
FIG. 5.
FIG. 5.
ORF33 is not required for MHV-68 early or late gene expression. ORF33 is not required for viral early or late gene expression. Firefly luciferase reporter constructs driven by ORF57 (A), ORF26 (B), or ORF65 (C) promoter were individually transfected into 293T cells. At 12 h posttransfection, the cells were infected with WT BAC, 33STOP, or 33STOP.R viruses at 0.1 infectious unit/cell. The firefly luciferase activity was measured at 24 h postinfection and was normalized against the Renilla luciferase internal control. The fold activation was calculated by comparing the normalized value of infected sample to that obtained from uninfected sample. Error bars represent standard deviations from three independent experiments.
FIG. 6.
FIG. 6.
ORF33 is required for maturation of MHV-68 virions in the cytoplasm. 293T cells were transfected with WT BAC plus pCMV-HA (A, B, and C), 33STOP plus pCMV-HA (D, E, and F), or 33STOP plus pHA-33 (G, H, I, and J). Approximately 70-nm thin sections were examined at 4 days posttransfection by transmission electron microscopy. Putative A capsid (a), B capsid (b), and C capsid (c) were found in WT BAC plus pCMV-HA, 33STOP plus pCMV-HA and 33STOP plus pHA-33-transfected cell nuclei (Nu) (A, D, G, and H). Primary enveloped virions of 33STOP (▾)were observed in the perinuclear space (E). In the cytoplasm (Cy), viral particles, including enveloped-tegumented virions (→) were observed in WT BAC plus pCMV-HA and 33STOP plus pHA-33 transfectants (B and I), but not in 33STOP plus pCMV-HA transfectant. Partially tegumented capsids (▴) were identified in 33STOP plus pCMV-HA transfectant (F). In the extracellular space, WT BAC plus pCMV-HA and 33STOP plus pHA-33 transfectants contained virions associated with cellular plasma membrane (V) (C and J), whereas no identifiable virions are found in 33STOP plus pCMV-HA transfectant.
FIG. 7.
FIG. 7.
Protein compositions of WT BAC, 33STOP, and complemented 33STOP particles. Viral particles were isolated from 293T cells cotransfected with WT BAC plus pCMV-HA, 33STOP plus pCMV-HA, or 33STOP plus pHA-33 at 4 days posttransfection. Virion proteins of WT BAC, 33STOP, and 33STOP plus HA-33 particles were separated by SDS-PAGE and examined by Western blotting, using polyclonal anti-MHV-68 serum (A), polyclonal antibodies to gB, ORF26, ORF65, ORF33, ORF45 or ORF52, or monoclonal anti-β-actin antibody (B).
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