Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus

Abstract

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.

Keywords: CRISPR/Cas9 genome editing; PrV; VAPA; VAPB; herpesvirus; nuclear egress; pseudorabies virus; vesicle-associated membrane protein associated protein.

Figure 1

CRISPR/Cas9 genome editing of VAPA- and VAPB-encoding genes in RK13 cells. Shown are the wildtype sequences (WT) and the deletions uncovered in the respective genes of the selected knockout cells (KO). In RK13-VAPA KO cells, 41 nt were deleted in the VAPA ORF (A), while 50 nt and additional 9 nt were removed in the VAPB coding region in RK13-VAPB KO (B). In both single KO cell lines, the alleles comprise identical changes. VAPA in RK13-VAPA/B double knockout (DKO) contained deletions of 15 nt (KO-I) or 1 nt (KO-II) (C), while the complete exon 2 in VAPB is missing (D). Due to the large deletion identical regions are indicated by black bars and the deleted sequence is represented by a thin line.

Figure 2

Immunoblots showing VAPA and VAPB in RK13, Vero and HeLa cells and the absence of VAPA and/or VAPB in the corresponding RK13 KO and DKO cell lines. Proteins in lysates of RK13, Vero and HeLa cells (A) or RK13 and the single as well as the double VAP KO cell lines (B) were separated on SDS-10 % polyacrylamide gels, and parallel blots were incubated with polyclonal sera against VAPA or VAPB. Masses of marker proteins are given on the left in kDa. As loading control, parallel (A) or the same blots were (re-)probed with anti-α-tubulin.

Figure 3

Actin organization is perturbed in RK13-VAPA/B DKO cells. RK13, RK13-VAPA KO, RK13-VAPB KO and RK13-VAPA/B DKO cells were fixed with 4% paraformaldehyde and cytoskeletal actin was stained with fluorophore-conjugated phalloidin (green). A loss of actin stress fibers and an accumulation of actin comets is obvious in RK13-VAPA/B DKO as reported for HeLa cells lacking VAPA/B [55]. Nuclei were counterstained with DAPI (blue). Shown are representative images. Scale bar = 20 µm.

Figure 4

Growth properties of PrV-Ka derived from parental RK13, VAP single and double KO cells. RK13, RK13-VAPA KO, RK13-VAPB KO and RK13-VAPA/B DKO were infected with PrV-Ka at an MOI of 5 (A) or 0.05 (B) and harvested after 24 or 48 h. Shown are mean values of progeny virus titers in plaque forming units per milliliter (pfu/ml) of five replicates with the corresponding standard deviation.

Figure 5

Ultrastructural analysis of PrV-Ka infected RK13, VAP KO and DKO cells. (A) RK13, (B) RK13-VAPA KO, (C) RK13-VAPB KO and (D) RK13-VAPA/B DKO cells were infected with PrV-Ka (MOI 1) and processed for electron microscopy 14 h post infection. Shown are presentative images. N: nucleus, C: cytoplasm.