Epstein-Barr virus that lacks glycoprotein gN is impaired in assembly and infection

Abstract

The Epstein-Barr virus (EBV) glycoproteins N and M (gN and gM) are encoded by the BLRF1 and BBRF3 genes. To examine the function of the EBV gN-gM complex, recombinant virus was constructed in which the BLRF1 gene was interrupted with a neomycin resistance cassette. Recombinant virus lacked not only gN but also detectable gM. A significant proportion of the recombinant virus capsids remained associated with condensed chromatin in the nucleus of virus-producing cells, and cytoplasmic vesicles containing enveloped virus were scarce. Virus egress was impaired, and sedimentation analysis revealed that the majority of the virus that was released lacked a complete envelope. The small amount of virus that could bind to cells was also impaired in infectivity at a step following fusion. These data are consistent with the hypothesis that the predicted 78-amino-acid cytoplasmic tail of gM, which is highly charged and rich in prolines, interacts with the virion tegument. It is proposed that this interaction is important both for association of capsids with cell membrane to assemble and release enveloped particles and for dissociation of the capsid from the membrane of the newly infected cell on its way to the cell nucleus. The phenotype of EBV lacking the gN-gM complex is more striking than that of most alphaherpesviruses lacking the same complex but resembles in many respects the phenotype of pseudorabies virus lacking glycoproteins gM, gE, and gI. Since EBV does not encode homologs for gE and gI, this suggests that functions that may have some redundancy in alphaherpesviruses have been concentrated in fewer proteins in EBV.

FIG. 1

(A) Diagram of the positions of the EcoRI and HindIII sites, numbered according to the B95-8 sequence, surrounding the EcoRI G fragment of Akata DNA targeted for homologous recombination. The boxes above indicate the position of the EcoRI G fragment used as a probe and the insertion of the Neo^r gene at bp 88598. Shown below are the sizes of the fragments expected from DNA from cells harboring wild-type episomes, a mixture of wild-type and recombinant episomes, or pure recombinant episomes after digestion with HindIII and probing with the EcoRI G fragment. (B) Southern blot analysis of DNA extracted from Akata cells harboring wild-type episomes (Wt), a parental clone of Akata cells harboring a mixture of wild-type and recombinant episomes (Wt+Rc), or a clone derived from the parental clone that contains only recombinant episomes (Rc). DNA was digested with HindIII, and the two identical halves of the membrane were cut apart and probed either with the EcoRI G fragment of EBV or the XmnI/HincII Neo^r fragment as indicated. The sizes are indicated in kilobases by arrows.

FIG. 2

Electrophoretic analysis in 9 to 18% polyacrylamide of proteins immunoprecipitated from Akata cells harboring wild-type or recombinant episomes. The cells were induced with anti-immunoglobulin antibody and labeled with [³H]glucosamine. Proteins were immunoprecipitated by anti-gL (αgL), which immunoprecipitates the EBV gH-gL complex of gH (85 kDa), gL (25 kDa), and gp42, by anti-gN (αgN), which immunoprecipitates the 15-kDa gN and the three 113-, 84-, and 48-kDa species of gM, by anti-gM (αM), and by preimmune antibody (PB). The sizes are indicated on the right in kilodaltons.

FIG. 3

Electron micrographs of induced Akata cells producing wild-type virus showing virus in the nucleus (A), in the cytoplasm (B and C), and extracellular virus (D). The arrows indicate enveloped virus and enveloped virus in vesicles in the cytoplasm.

FIG. 4

Electron micrographs of induced Akata cells producing recombinant virus lacking gN showing virus associated with chromatin in the nucleus (A to C) or in the cytoplasm (D). The arrows in panels A to C indicate virus in the condensed chromatin in the nucleus. The arrows in panel D indicate nonenveloped virus in the cytoplasm.

FIG. 5

Southern blot of Gardella gel analysis of the amount of virion DNA that bound to EBV-negative Akata cells. The amounts of virion DNA in wild-type (Wt) or one of two independently isolated recombinant viruses (Rc1 and Rc2) that were added were equilibrated. Scanning of exposures of Southern blots within the linear range indicated that at least 10-fold more of the wild type than the recombinant virion DNA bound to cells.

FIG. 6

Southern blot of Gardella gel analyses of the amounts of wild-type (Wt) and recombinant viruses (Rc) that bound to EBV-negative Akata cells alone or in combination. The starting concentration of each virus stock was equilibrated for virus DNA content. Scanning of the Southern blot at exposures within the linear range indicated that the amount of virion DNA bound in the mixtures of the different viruses was in each case what would have been expected from a simple addition of each virus. The two panels represent duplicate analyses.

FIG. 7

Sedimentation profiles in 24 to 42% Nycodenz of wild-type virus (A) and recombinant virus (B). The scale of the first 15 fractions of panel B are enlarged in panel C. Samples of each fraction were measured by slot blot analysis for the relative amounts of virion DNA and by Gardella gel analysis for the relative amounts of virion DNA that could bind to EBV-negative Akata cells. Equal amounts of virion DNA were loaded for each virus. Variables: ▴, refractive index; ●, virion DNA; ○, virus binding.

FIG. 8

Comparison of the infectivity of wild-type (Wt) and recombinant (Rc) viruses after attachment of equal amounts of virion DNA. (Upper panel) Southern blot of Gardella gel analysis, in duplicate, of amounts of virion DNA bound to EBV-negative Akata cells after addition of different dilutions of wild-type and recombinant viruses. (Lower panel) Infectivity of the same dilutions of virus measured by Western blot analysis for the EBV latent protein EBNA1.

FIG. 9

Western blot analysis for EBNA1 expression in T-cell-depleted human mononuclear cells infected in the absence or presence of polyethylene glycol (PEG) with wild-type Akata virus (Wt), recombinant virus lacking gN (Rc/gN), or recombinant virus lacking gp42 (Rc/gp42).

FIG. 10

Binding and infectivity of equal amounts of recombinant viruses that had been produced from AGS cells (Rc) or from AGS expressing gN from an integrated plasmid (Complemented Rc). (Upper panel) Southern blot of Gardella gel analysis of amounts of dilutions of virion DNA bound to EBV-negative Akata cells. (Lower panel) Infectivity of dilutions of virus measured by Western blot analysis for the EBV latent protein EBNA1.

FIG. 11

Comparison of replication of wild-type virus and virus lacking the gN-gM complex. (A) Wild-type virus. Wild-type virus buds through the inner nuclear membrane and either follows the default exocytic pathway to the cell surface or undergoes a second step of de-envelopment and re-envelopment. Enveloped particles bind to a new cell and fuse with the cell membrane, and the capsid moves away from the membrane to the nucleus. (B) Virus lacking the gN-gM complex. Many gN-null capsids associate with condensed chromatin. A few appear as enveloped particles in vesicles, and a significant amount of the virus is released without an intact envelope. Virus that remains able to bind to new cells is impaired in infectivity at a step following fusion, perhaps involving movement of capsids away from the cell membrane to the nucleus.