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. 2015 May;89(10):5288-97.
doi: 10.1128/JVI.00079-15. Epub 2015 Feb 25.

Gammaherpesvirus Tegument Protein ORF33 Is Associated With Intranuclear Capsids at an Early Stage of the Tegumentation Process

Sheng Shen  1 , Xing Jia  2 , Haitao Guo  1 , Hongyu Deng  3
Affiliations

Gammaherpesvirus Tegument Protein ORF33 Is Associated With Intranuclear Capsids at an Early Stage of the Tegumentation Process

Sheng Shen et al. J Virol. 2015 May.

Abstract

Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in the nuclear egress of capsids into the cytoplasm, especially for the two human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to a lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study the de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remains unclear how ORF33 is incorporated into virions. In this study, we first show that the endogenous ORF33 protein colocalizes with capsid proteins at discrete areas in the nucleus during viral infection. Cosedimentation analysis as well as an immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. An immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we show that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 to be a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner.

Importance: Morphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although the process of capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between the capsid and the envelope in a herpesvirus virion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions in both the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we show that ORF33 is associated with intranuclear capsids prior to primary envelopment and identify novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids at an early stage of the virion maturation process for herpesviruses.

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Figures

FIG 1
FIG 1
MHV-68 ORF33 accumulates in the nucleus and colocalizes with capsid proteins during virus infection. (A) Verification of the specificity of the anti-ORF33 monoclonal antibody. Vero and BHK-21 cells were transfected with pHA-33, the WT BAC, or the 33Stop BAC. At 36 h posttransfection, cells were lysed and subjected to Western blotting with the anti-ORF33 monoclonal antibody, a polyclonal antibody against ORF26, or a monoclonal antibody specific for β-actin. Lysates from mock-transfected cells were used as a negative control. (B) ORF33 colocalizes with capsid proteins during viral infection. Vero cells were infected with MHV-68 at an MOI of 3. At 12 and 24 h postinfection, cells were fixed and subjected to an indirect immunofluorescence assay using the anti-ORF33 monoclonal antibody and a rabbit polyclonal antibody against ORF65 or ORF26. The anti-ORF33 antibody was detected by use of an Alexa Fluor 568-conjugated secondary antibody (red channel). The anti-ORF65 and amti-ORF26 antibodies were detected by use of an Alexa Fluor 488-conjugated secondary antibody (green channel). Arrows, cytoplasmically localized ORF33.
FIG 2
FIG 2
Cosedimentation of ORF33 with both nuclear and cytoplasmic capsids. (A) BHK-21 cells were infected with MHV-68 at an MOI of 5. At 12 h postinfection, the cells were harvested and separated into nuclear (Nu) and cytoplasmic (Cy) fractions, as described in Materials and Methods. A small portion of the two fractions was analyzed by immunoblotting with antibodies against lamin A/C or caspase 3. (B) Nuclear and cytoplasmic capsids were first pelleted through a 30% sucrose cushion prior to 20 to 50% (wt/vol) sucrose gradient sedimentation. Fractions were collected, and proteins were concentrated and separated on SDS–12% polyacrylamide gels prior to immunoblotting analyses with antibodies against ORF33 or capsid protein ORF26.
FIG 3
FIG 3
Immunoprecipitation of both nuclear and cytoplasmic capsids by the anti-ORF33 antibody. (A) BHK-21 cells were infected with MHV-68 at an MOI of 5 for 12 h. Nuclear and cytoplasmic fractions were separated as described in Materials and Methods and analyzed as described in the legend to Fig. 2A. (B) Nuclear and cytoplasmic fractions were incubated with the mouse anti-ORF33 monoclonal antibody or mouse IgG. Each precipitated sample pulled down with the anti-ORF33 antibody was divided into two parts. One part was analyzed by Western blotting to detect viral proteins using antibodies against ORF33, ORF65, or ORF26 (lanes 2 and 4). The other part was treated with trypsin for 1 h in the presence of 1% Triton X-100 prior to Western blotting analyses (lanes 5 and 6). Precipitates pulled down by mouse IgG were also analyzed by Western blotting (lanes 1 and 3). Cy, cytoplasmic extract; Nu, nuclear extract; IP, immunoprecipitation.
FIG 4
FIG 4
ORF33 is associated with nuclear capsids prior to primary envelopment as well as with mature virions in the cytoplasm. (A to F) BHK-21 cells were infected with WT MHV-68 at an MOI of 5 and analyzed 11 h later by immunoelectron microscopy with the anti-ORF33 monoclonal antibody, followed by a secondary antibody conjugated with 10-nm gold particles. Gold particles were detected both on or adjacent to capsids in the nucleus (Nu) (A to D) and on or adjacent to virions in the cytoplasm (Cyto) (E and F). (G and H) BHK-21 cells transfected with the 33Stop BAC were used as a negative-control sample. No gold particles were detected on the capsids. Black arrows, gold particles closely associated with capsids or virions; white arrows, gold particles adjacent to capsids or virions; arrowheads, free gold particles.
FIG 5
FIG 5
Immunogold labeling of ORF33-rich regions in the nucleus and cytoplasm. Samples were treated and viewed as described in the legend to Fig. 4. Arrows, nuclear capsids; black arrowheads, ORF33-rich nuclear areas; white arrowheads, ORF33-rich cytoplasmic area.
FIG 6
FIG 6
ORF33 interacts with capsid proteins ORF25 and ORF26. 293T cells were cotransfected with expression plasmids, as indicated. At 48 h posttransfection, whole-cell lysates (WCL) were prepared, and the expression of each protein was examined by Western blotting. The whole-cell lysates were further subjected to immunoprecipitation with anti-Flag M2 agarose and analyzed by Western blotting using anti-HA, anti-FLAG, or anti-ORF33 antibodies. Arrowheads, antibody heavy and light chains.
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