Asna1/TRC40 that mediates membrane insertion of tail-anchored proteins is required for efficient release of Herpes simplex virus 1 virions

HeLa (ATCC CCL-2) and Vero cells (ATCC CRL-1587) were grown in DMEM containing 10 % FCS. Yeast 2-hybrid (Y2H) analysis was done as described [14]. The UL34, UL45, UL56 and US9 genes previously cloned into the entry vector pDONR207 [15] were transferred into the Gateway compatible Y2H bait vector pGBKT7-DBD and/or the mammalian expression vector pCR3-N-myc according to the manufacturer’s protocol (Invitrogen). The human Asna1/TRC40 gene previously cloned into the pDONR223 vector was transferred to the Gateway compatible Y2H prey vector pGADT7-AD according to the manufacturer’s protocol (Invitrogen).

HSV1(F) (provided by B. Roizman, University of Chicago, USA) was used for infection experiments. The strain HSV1(17+)lox (provided by B. Sodeik, Hannover Medical School, Germany) was used as PCR template. HSV1 propagation and virus growth curves were performed as described [14]. To monitor infection, Vero cells were infected with HSV1(F) at the indicated MOI. Cell lysates were prepared at the indicated times post infection and analysed by Western blotting using primary antibodies to the immediate early proteins ICP0 (anti-ICP0, Santa Cruz) and ICP27 (anti-ICP27, Virusys), to the early protein gB (anti-Glykoprotein B, Santa Cruz) and to the late proteins VP5 (anti-ICP5 (VP5), Abcam) and pUL34 [9] followed by secondary antibodies conjugated to POX. Antibodies specific to β-actin (Abcam) were used as control.

Indirect immunofluorescence (IF) analysis of transfected or infected cells was done as described [14]. For plasmid transfection, the Effectene Transfection Reagent was used. For virus infection, HeLa cells were infected at the indicated MOI. In infected cells, binding of antibodies to the HSV1 Fc-receptor like proteins gE/gI was blocked overnight at 4 °C with human IgG (200 μg/ml) and 10 % FCS in PBS [16]. The mouse monoclonal antibodies anti-myc [9E10] (Santa Cruz), anti-ICP8 (provided by R. Heilbronn, Charité Universitätsmedizin Berlin CCM, Berlin, Germany), anti-Asna1/TRC40 ([M03], Klon 2H3 Abnova) and rabbit polyclonal antibodies anti-pUL34 [9], anti-Calreticulin (Sigma), and anti-Giantin (Abcam) were used as primary reagents. Goat anti-rabbit or anti-mouse antibodies coupled to Alexa488 or Alexa594 (all Invitrogen) were used as secondary reagents. Cells were examined using a Leica confocal laser scanning microscopes TCS SP5 and LSM710. Images were recorded using the Leica Application Suite AF6000 Software and processed using Adobe Photoshop.

Gene silencing was essentially done as described [17]. Briefly, siRNAs (20 nM; GE Dharmacon), 150 μl of HBSS, and 1,5 μl of transfection reagent were mixed and added to HeLa cells in DMEM with 5 % FCS seeded in 12-well plates. Efficiency of RNAi was monitored by Western blotting using mouse monoclonal antibodies to Asna1/TRC40 ([M03], Klon 2H3 Abnova) and polyclonal goat anti-Lamin B (Santa Cruz) to control for loading. The siRNA duplexes used for Asna1/TRC40 knockdown and as controls (Ctrl) are shown in Table 1. Viral infection was generally performed 48 h (h) after siRNA treatment. Infectious virions were quantified by removing aliquots of medium and of infected cells at various time points followed by plaque assay on Vero cells [14]. To determine genome copy/pfu ratios of virions released from cells, HeLa cells were treated with Asna1/TRC40 specific or ctrl siRNA followed by infection with HSV1(F) for 30 h. Realtime quantitative PCR using HSV1 specific primers were used to determine the genome copies, plaque assays were performed as described [14].

HSV1 encodes three TA proteins, called pUL34, pUL56 and pUS9. Hydrophobicity plots show that pUL34, an essential protein conserved throughout the herpesviral family, contains a transmembrane domain (TMD) between residues 252–272 required for nuclear egress (Fig. 1a; [9]). Two other TA proteins, pUL56 and pUS9, that are non-essential and specific for alpha-herpesviruses, carry a TMD between residues 211–231 and 69–89, respectively (Fig. 1a).

To determine whether pUL34, pUL56 and pUS9 interact with Asna1/TRC40, the yeast 2-hybrid (Y2H) system was applied. Asna1/TRC40 fused to the Gal4 activation domain (AD) was tested for interaction with pUL34, pUL56 and pUS9 fused to the Gal4 DNA-binding domain (DBD). pUL45, that carries an N-terminal TMD co-translationally integrated into membranes by an Asna1/TRC40-independent mechanism, was used as control. Interaction of proteins was reported by growth of yeast cells on selective media. While DBD-pUL34, -pUL56 and -pUS9 co-expressed with AD-Asna1/TRC40 allowed for growth of yeast cells, this was not the case for co-expression of DBD-pUL45 and AD-Asna1/TRC40 (Fig. 1b). We thus conclude that all three TA proteins of HSV1 specifically interacted with Asna1/TRC40 supporting its function in posttranslational membrane insertion of these viral proteins.

To analyse the subcellular distribution of Asna1/TRC40 in presence and absence of HSV1 infection, HeLa cells were mock treated or infected with HSV1(F) for 12 h and subsequently processed for IF. Infected cells were readily identified based on their marginalized chromatin as revealed by DAPI staining (Fig. 1c). Both in noninfected and infected cells, Asna1/TRC40 showed a pancellular distribution and significantly co-localized with the ER marker Calreticulin (Fig. 1c) suggesting that its distribution is essentially unaltered during HSV1 infection (Fig. 1c).

In absence of other viral proteins, pUL34 is targeted to the ER and the nuclear periphery, whereas pUL56 and pUS9 are located to the trans Golgi network (TGN). To determine whether Asna1/TRC40 is required for proper membrane targeting of the HSV1 TA proteins, HeLa cells were transfected for 48 h with Asna1/TRC40 specific or control (ctrl) siRNA. Knockdown of Asna1/TRC40 was highly efficient as shown by Western blotting (Fig. 2a). Interestingly, depletion of Asna1/TRC40 did not affect cell viability of Hela cells (data not shown).

RNAi treated cells were then transfected with plasmids encoding myc-tagged TA proteins and 20 h later analysed by IF using monoclonal anti-myc antibodies. Calreticulin or Giantin were used as markers of the ER and the TGN, respectively. pUL34 showed a reticular subcellular distribution and co-localized with the ER marker Calreticulin consistent with its localization to the ER and the nuclear periphery whether the cells were treated with Asna1/TRC40 specific or ctrl siRNA (Fig. 2b, left panel). pUL56 and pUS9 both located to the TGN as indicated by their co-localization with the TGN marker Giantin (Fig. 2b, middle and right panel). In Asna1 depleted cells, a certain amount of pUS9 was found in a perinuclear region suggesting that membrane insertion of pUS9 is influenced by the absence of Asna1. To summarize, all HSV1 TA proteins seemed to efficiently reach their target membranes irrespective of the presence or absence of Asna1/TRC40.

To determine whether Asna1/TRC40 is required for the herpesviral life cycle, Asna1/TRC40 knockdown was performed and monitored as shown before (Fig. 2a). Then, siRNA treated HeLa cells were infected with HSV1 at an MOI of 0.5 for 4 h (Fig. 3a). ICP0 expression was analysed as indirect means of viral entry. IF analysis revealed that 20 % and 19 % of the cells treated with ctrl- and Asna1/TRC40-specific siRNAs, respectively, were infected by HSV1(F).

A time course experiment to detect viral proteins of all kinetic classes was performed. HeLa cells were first treated with Asna1/TRC40 or ctrl siRNA for 48 h and subsequently infected with HSV1 at an MOI of 1. Cell lysates were prepared at the indicated time-points and probed with antibodies specific to the immediate early regulators ICP0 and ICP27, to glycoprotein gB, to the nuclear egress protein pUL34 and the major capsid protein ICP5 (VP5). β-Actin-specific antibodies were used to control for equal loading of cell samples (Fig. 3b). The transcriptional regulators ICP0 and ICP27 were detected 2 h post infection (h.p.i.), glycoprotein gB and the nuclear egress protein pUL34 appeared 6 h.p.i., while the major capsid protein ICP5 was detected 8 h.p.i.. Taken together, we found that Asna1/TRC40 is not required for virion entry and overall viral gene expression (Fig. 3b).

HSV1 pUL34 is essential with a conserved function in nuclear egress of capsids. To determine whether Asna1/TRC40 is required for pUL34 biogenesis in the viral context, RNAi was performed as described (Figs. 2a and 4a). Subsequently, HeLa cells were infected with HSV1 at an MOI of 1 for 12 h and subsequently processed for IF (Fig. 4b and c). Antibodies specific to pUL34 (Fig. 4b) and Lamin B (Fig. 4c) showed that both proteins were exclusively located to the nuclear envelope. Furthermore, intranuclear replication centers formed normally, as revealed by ICP8-specific antibodies (Fig. 4b and c). Thus, we conclude that pUL34 membrane insertion and targeting to the INM, a prerequisite for NEC formation and capsid nuclear egress, proceeds normally in absence of Asna1/TRC40.

To determine the overall effect of Asna1/TRC40 knockdown on the outcome of an HSV1 infection, Asna1/TRC40 depleted HeLa cells or ctrl cells were infected with HSV1 at an MOI of 0.1. At the indicated time points, medium and infected cells were harvested separately and analysed for the presence of infectious virions using plaque assays. Equal amounts of infectious virions were formed and remained cell-associated whether cells were treated with Asna1/TRC40 specific or ctrl siRNA (Fig. 5a). In contrast, about 10 times less infectious virions were released to the culture medium upon Asna1/TRC40 depletion. To determine the genome copy/pfu ratio of virus released 30 h.p.i. from the infected cells, realtime quantitative PCR was performed. Virions released from Asna1 siRNA treated cells showed reduced amounts of genomes as well as plaque forming units compared to the ctrl treated cells. In either case however, their genome copy/pfu ratio was comparable indicating that virions released to the extracellular milieu were similar in infectivity (Fig. 5b). Thus, although not essential for cellular growth and formation of infectious virions, Asna1/TRC40 is required for release of mature virions from the cells at a late step of virus infection and thus for efficient HSV1 propagation.