The Epstein-Barr virus SM protein is functionally similar to ICP27 from herpes simplex virus in viral infections

Abstract

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm to increase the cytoplasmic accumulation of viral late mRNAs. ICP27 homologs have been identified in each of the herpesvirus subfamilies, and accumulating evidence indicates that homologs from the gammaherpesvirus subfamily function similarly to ICP27. In particular, the Epstein-Barr virus (EBV) SM protein posttranscriptionally regulates gene expression, binds RNA in vitro and in vivo, and shuttles between the nucleus and cytoplasm. To determine if these two proteins function through a common mechanism, the ability of EBV SM to complement the growth defect of an HSV-1 ICP27-null virus was examined in a transient-expression assay. ICP27 stimulated the growth of the null mutant more efficiently than did SM, but the ability of SM to compensate for the ICP27 defects suggests conservation of common functions. To assay for complementation in the context of a viral infection, the growth properties of an HSV recombinant expressing SM in an ICP27-null background were analyzed. SM stimulated growth of the recombinant, although this growth was reduced by comparison to that of an ICP27-expressing virus. By contrast, an HSV recombinant expressing an SM mutant allele defective for transactivation activity and nucleocytoplasmic shuttling did not grow at all. These results suggest that SM and ICP27 may regulate gene expression through a common pathway that is evolutionarily conserved in herpesviruses.

FIG. 1.

Evolutionary relationships among HSV-1 ICP27 homologs. An unrooted phylogenetic tree was generated from an amino acid alignment of the conserved carboxy-terminal portion of sequences corresponding to ICP27 and its homologs (HSV-1 ICP27 amino acid residues 264 to 508). The sequences represent viruses from the α (HSV-1, HSV-2, BHV, EHV, PRV, MDV, and VZV), β (CMV and HHV-6), and γ (EBV, HVS, and KSHV) herpesvirus subfamilies. Criteria for inclusion in the analysis were BlastP homology to the HSV-1 strain 17 ICP27 protein (HSV-2, CMV, MDV, PRV, and VZV) or to the CMV UL69 protein (EBV, KSHV, and HVS). Bootstrap values for each branch are shown at the node as the number of occurrences in 100 bootstrap test trees.

FIG. 2.

Complementation of vBSΔ27 growth by HSV-1 ICP27 homologs. Vero cells were transiently transfected with CMV promoter-based expression plasmids encoding HSV-1 ICP27, EBV SM, EBV SMLRRΔ, HCMV UL69, VZV ORF4, or, as a control, the empty parental plasmid. At 24 h posttransfection, the cells were infected with vBSΔ27, and the transfected-infected cells were collected at 24 h postinfection. To normalize for transfection efficiency in individual experiments, a green fluorescent protein expression plasmid was included in all transfection reactions. To make comparisons between experiments, all experiments included a wild-type ICP27 expression plasmid, and data were normalized to complementation by wild-type ICP27. (A) Complementation of viral growth was measured by plaque assay on 2-2 cells. The y axis denotes virus growth on a logarithmic scale, and the x axis denotes the transiently expressed protein (HSV-1 ICP27, EBV SM, EBV SMLRRΔ, HCMV UL69, or VZV ORF4). If any viral growth was stimulated in cells transfected with the empty control vector, this value was subtracted from growth stimulated in the presence of the viral proteins. The dashed line indicates the limit of detection of the assay. The data represent the averages of at least three independent experiments. (B) The expression of HSV ICP27 or EBV SM protein synthesized in complementation assays was measured by immunoblotting with antiserum specific for ICP27 (Clu38), SM (α-SM), UL69 (α-HA), or ORF4 (α-VZV ORF4). At the top of each lane, the transfected plasmids are labeled as C (control), 27 (pCMV27), SM (pCMVSM), EBV SMLRRΔ (pVR114), UL69 (pCGNUL69), or ORF4 (pVZV4).

FIG. 3.

Construction of EBV SM-expressing recombinant herpes simplex viruses. To analyze growth complementation of an ICP27 deletion mutant by the EBV SM protein in the context of a viral infection, a recombinant vBSΔ27-based virus containing the EBV SM ORF in the tk locus was constructed. The EBV SM ORF was placed under the control of the ICP27 promoter and cloned into a plasmid containing the complete tk gene to provide flanking homology with tk (p27PSM/TK fragment, panel C; expanded tk locus, panel B). In the resulting targeting plasmid, the SM insertion interrupts the tk ORF. Recombinant viruses were constructed in a vBSΔ27 background (A) by cotransfection of a linear p27PSM/TK fragment with infectious vBSΔ27 nucleocapsids into the ICP27-complementing cell line 2-2. Recombinants from two independent cotransfections were selected by plaquing on 2-2 cells in the presence of 5-bromodeoxycytidine. vSM/TK-Δ27 recombinants were screened by PCR (D) for the retention of lacZ coding sequence at the ICP27 locus (top panel). Individual isolates of plaques PCR positive for SM are labeled across the top of the lanes. The lane labeled Δ27 is a control reaction from a vBSΔ27 plaque. Potential vSM/TK-Δ27 recombinants were screened by PCR for the presence of SM coding sequence and simultaneously for the absence of tk coding sequence with a three-primer reaction described in detail in Materials and Methods (bottom panel). Individual isolates of PCR-positive viruses are labeled across the top of the lanes. The lane labeled MP is a control PCR from a reaction containing two plasmids, pTK-29/-19 (tk containing) and p27PSM/TK (SM containing), and the lane labeled Δ27 is a control reaction from a vBSΔ27 plaque. For Southern hybridization of recombinant virus genomic DNA, cytoplasmic DNA from vBSΔ27- or vSM/TK-Δ27-infected cells was digested with SalI and hybridized with a random-primed probe complementary to tk as described in Materials and Methods (E). The locations of the probe and the hybridization signals (B and C) diagnostic for wild-type tk (5,948 bp) and SM-replaced tk (5,374 and 2,474 bp) are indicated. Homology between the SM-replaced tk locus and the wild-type tk locus is indicated by shading.

FIG. 4.

Growth of vSM/TKΔ27in low- and high-MOI infections. (A) Vero cells were infected with vSM/TK-Δ27 (SM/TK) or v27/TK-Δ27 (27/TK) at an MOI of 5 PFU/cell, and the infected cells were collected at the indicated times postinfection. The resulting viral yields were determined on 2-2 cells. Data points represent the averages of three independent infections, each performed in duplicate. (B) Vero cells were infected with two independent isolates of vSM/TK-Δ27 (SM/TK.B1 or SM/TK.A16), v27/TK-Δ27 (27/TK), or vBSΔ27 (Δ27) at an MOI of 0.1 PFU/cell, and the infected cells were collected at the indicated times postinfection. The resulting viral yields were determined on 2-2 cells. Data points represent the averages of two independent infections, each performed in duplicate.

FIG. 5.

Analysis of viral spread by indirect immunofluorescence. Vero cells were seeded onto glass coverslips and infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), v27/TK-Δ27 (27/TK-Δ27), or HSV-1 KOS (wild type) at an MOI of 0.01 PFU/cell. At the indicated times postinfection, cells on the coverslips were fixed and processed for indirect immunofluorescence with a monoclonal antibody reactive with the immediate-early ICP4 protein H1114. Representative fields of cells are shown for each time point. Two representative fields are presented for vSM/TK-Δ27 at 24, 48, and 72 h postinfection to illustrate the heterogeneity of these infections. U, upper panels; L, lower panels.

FIG. 6.

Growth of vSMLRRΔ/TK in low-MOI infections. Vero cells were infected with vSMLRRΔ/TK (SMLRRΔ/TK), vSM/TK-Δ27 (SM/TK), v27/TK-Δ27 (27/TK), or vBSΔ27 (Δ27) at an MOI of 0.1 PFU/cell, and the infected cells were collected at the indicated times postinfection. The resulting viral yields were determined on 2-2 cells. Data points represent the averages of three independent infections, each performed in duplicate.

FIG. 7.

Accumulation of viral proteins in vSM/TK-Δ27-infected cells. Vero cells were mock infected (uninfected) or infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), or v27/TK-Δ27 (27/TK-Δ27) at an MOI of 0.1 PFU/cell, and lysates of infected cells were prepared at the indicated times (hours) postinfection. The relative abundance of the indicated HSV proteins was determined by immunoblotting with antibodies reactive with UL38, gC, VP16, ICP8, ICP4, EBV SM, and ICP27. These proteins are representative of all HSV kinetic classes (α, β, γ₁, and γ₂). The asterisks on the SM immunoblot of SM/TK-Δ27-infected cells denote a cross-reacting protein induced by viral infection that is also present in cells infected with vBSΔ27 and v27/TK-Δ27.

FIG. 8.

Viral DNA replication. Vero cells were either mock infected or infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), or v27/TK-Δ27 (27/TK-Δ27) at an MOI of 0.1 PFU/cell. Aliquots of infected cells were collected at the indicated times (hours) postinfection, and 10-fold dilutions (0.1× and 0.01×) were slot blotted onto nylon membranes. The DNAs were hybridized with a ³²P-labeled probe complementary to the ICP4 loci.

FIG. 9.

Synthesis of cellular proteins in vSM/TK-Δ27-infected cells. Vero cells were either mock infected (M) or infected with vSM/TK-Δ27 (SM/TK-Δ27), vBSΔ27 (Δ27), v27/TK-Δ27 (27/TK-Δ27), or wild-type HSV-1 KOS at an MOI of 10 PFU/cell. The cells were pulse-labeled with Tran³⁵S Label at 3, 7, and 11 h postinfection (hpi). Total-cell extracts were analyzed by SDS-PAGE, and labeled proteins were visualized by autoradiography. Arrows on the right identify viral proteins; solid circles on the left identify cellular proteins that exhibit a reduction in synthesis as vSM/TK-Δ27, v27/TK-Δ27, or wild-type HSV-1 infections progress, and asterisks indicate cellular proteins that exhibit reduced synthesis in infections where ICP27 is expressed but continue to be synthesized in vSM/TK-Δ27 infections.