RASCAL is a new human cytomegalovirus-encoded protein that localizes to the nuclear lamina and in cytoplasmic vesicles at late times postinfection

Abstract

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.

FIG. 1.

Genomic location of the c-ORF29 gene and in silico analysis of the RASCAL amino acid sequence. (A) Schematic map of the TB40-BAC4 (GenBank accession no. EF999921) and of the Towne-BAC (accession no. AY315197) genomic regions corresponding to nucleotides 9200 to 10200 (TB40/E) and 198750 to 199750 (Towne). The black horizontal line depicts the viral genome, with vertical lines positioned every 500 nucleotides. ORFs are represented by arrows pointing in the direction of transcription. (B) ClustalW2 alignment of RASCAL amino acid sequences from 14 human CMV strains. Viral genome accession numbers are as follows: TB40-BAC, EF999921; PH-BAC, AC146904; TR-BAC, AC146906; FIX-BAC, AC146907; 3157, GQ221974; 3301, GQ466044; HAN13, GQ221973; JP, GQ221975; Merlin, AY44689; AD169, X17403; HAN38, GQ396662; HAN20, GQ396663; Toledo-BAC, AC146905; and Towne-BAC, AC146851. Dots and dashes indicate identical and absent amino acids, respectively. The asterisks mark the specific amino acid predicted to be phosphorylated by PKA or PKC or to be N glycosylated (N-glyc). The black lines underscore the putative TMDs. (C) Kyte-Doolittle (K-D) hydropathy plot of RASCAL_TB40/E and RASCAL_Towne amino acid sequences, performed using a window size of 9 aa. The shaded boxes include the stretch of amino acids corresponding to the putative TMDs, while the boxes with the dashed borders include the two additional hydrophobic regions of more than 17 aa detected in RASCAL_Towne.

FIG. 2.

RASCAL expression in infected and transfected cells. (A, top) Schematic depiction of the primers used to amplify c-ORF29 by RT-PCR from TB40/E- or Towne/GFP-IE2-infected HF. The > and < symbols depict the direction of polymerization. The sequence of primer a > is complementary to nucleotides 1 to 13 of c-ORF29_TB40/E and of c-ORF29_Towne, the sequence of primer < b is complementary to nucleotides 277 to 291 of c-ORF29_TB40/E and of c-ORF29_Towne and to nucleotides 189 to 203 of US17, while the sequence of primer < c is complementary to nucleotides 515 to 528 of c-ORF29_Towne and nucleotides 427 to 440 of US17. (Bottom) RT-PCR analysis of c-ORF29 transcription in mock- or TB40/E-infected HF at the indicated times p.i., using primers a > and < b. Amplification of vimentin's cDNA was used as the PCR control. (B) RT-PCR analysis of c-ORF29 and of UL99 transcription in HF infected with TB40/E in the presence (+) or absence (−) of PFA (300 μg/ml). Amplification of β-actin's cDNA was used as the PCR control. (C) Immunoblot analysis results of RASCAL_TB40/E expression in protein extracts from HEK293T cells nontransfected or transfected with expression plasmids encoding RASCAL_TB40/E-GFP or RASCAL_TB40/E. The blot was incubated with an anti-RASCAL polyclonal Ab as described in Materials and Methods. Expected molecular masses were 10.6 kDa for RASCAL_TB40/E and 37.6 kDa for RASCAL_TB40/E-GFP. Asterisk, RASCAL_TB40/E; arrowhead, RASCAL_TB40/E-GFP. (D) Immunoblot analysis of RASCAL expression in protein extracts from mock-, TB40/E-, or Towne/GFP-IE2-infected HF and in HEK293T cells expressing RASCAL_TB40/E. The same membrane was incubated first with an anti-RASCAL polyclonal Ab (left) and subsequently with the preimmune serum (right) as described in Materials and Methods. Expected molecular masses were 10.6 kDa for RASCAL_TB40/E and 19.4 kDa for RASCAL_Towne. Asterisk, RASCAL_TB40/E; square, RASCAL_Towne.

FIG. 3.

RASCAL intracellular localization. Mock-infected (A to C) or Towne/GFP-IE2-infected HF (MOI of 5) (D to R) were harvested at the indicated times p.i. and were stained with affinity-purified rabbit anti-RASCAL polyclonal Abs followed by Alexa Fluor 594-conjugated goat anti-rabbit Abs. The signal emitted from the GFP-IE2 protein was further amplified with FITC-conjugated anti-IE1/IE2 Abs, and nuclear DNA was stained with Hoechst 33342. The arrows point at RASCAL accumulation at the nuclear rim, the arrowheads indicate the peculiar structures observed on the nuclear surface at late times p.i., and the asterisks mark the locations of the cytoplasmic RASCAL-positive vesicles. Original magnification, ×400.

FIG. 4.

RASCAL expression in the absence of viral DNA synthesis. HF infected with Towne/GFP-IE2 (MOI of 3) in the presence or absence of PFA (300 μg/ml) were harvested at the indicated times p.i. and stained with affinity-purified rabbit anti-RASCAL polyclonal Abs followed by Alexa Fluor 594-conjugated goat anti-rabbit Abs and FITC-conjugated anti IE1/IE2 Abs. Original magnification, ×400.

FIG. 5.

RASCAL colocalization with lamin B and with lamin A/C at the nuclear envelopes of infected cells. Confocal images of TB40/E-infected HF (MOI of 5) harvested at 72 hpi (A to C) or 96 hpi (D to L) and stained with affinity-purified rabbit anti-RASCAL polyclonal Abs followed by Alexa Fluor 594-conjugated goat anti-rabbit Abs (red), with monoclonal anti-lamin B Abs followed by FITC-conjugated anti-mouse Abs (green), or with monoclonal anti-lamin A/C Abs followed by FITC-conjugated anti-mouse Abs (green). Bar size, 10 μm.

FIG. 6.

RASCAL colocalization with lamin B but not lamin A/C in cytoplasmic vesicles. Confocal images of TB40/E-infected HF (MOI of 5) harvested at 96 hpi and stained with affinity-purified rabbit anti-RASCAL polyclonal Abs followed by Alexa Fluor 594-conjugated goat anti-rabbit Abs (red), with monoclonal anti-lamin B Abs followed by FITC-conjugated anti-mouse Abs (green) (A to F), or with monoclonal anti-lamin A/C Abs followed by FITC-conjugated anti-mouse Abs (green) (G to I). The area magnified in panels D to F is framed by a square box in panels A to C. Arrowheads indicate the points of close contact between the cytoplasmic vesicles and the nuclear lamina. Bar size, 5 μm.

FIG. 7.

Localization of UL50-HA, UL53-FLAG, and RASCAL_TB40/E in transfected cells. (A to D) Confocal images of HEK293T transiently transfected with expression plasmids encoding UL50-HA (A and B) or UL53-FLAG (C and D) and stained with mouse anti-HA Ab (A), mouse anti-FLAG Abs (C), or affinity-purified rabbit anti-RASCAL polyclonal Abs (B and D) followed by FITC-conjugated goat anti-mouse Abs (green) or Alexa Fluor 594-conjugated goat anti-rabbit Abs. (E to M) Confocal images of HEK293T coexpressing UL50-HA and UL53-FLAG (E to F), UL53-FLAG and RASCAL_TB40/E (H to J), or UL50-HA and RASCAL_TB40/E (K to M). Cells were stained with rabbit anti-HA and mouse anti-FLAG Abs followed by Alexa Fluor 594 goat anti-rabbit Abs (green) and FITC-conjugated goat anti-mouse Abs (red) (E to G), with mouse anti-FLAG and rabbit anti-RASCAL Abs followed by FITC-conjugated goat anti-mouse Abs (green) and Alexa Fluor 594-conjugated goat anti-rabbit Ab (red) (H to J), or with mouse anti-HA and rabbit anti-RASCAL Abs followed by FITC-conjugated goat anti-mouse Abs (green) and Alexa Fluor 594-conjugated goat anti-rabbit Abs (red) (K to M). Bar size, 10 μm.

FIG. 8.

Determination of RASCAL's interaction with UL50 by co-IP. (A) Immunoblot analysis of protein extracts from HEK293T cells transiently transfected with LNCX or cotransfected with L-RASCAL_TB40/E and LNCX UL50-HA. Proteins were denatured in 3% SDS lysis buffer (lysate) or were subjected to co-IP with anti-HA Abs (left) or with anti-RASCAL Abs (right) prior to separation on 10% (UL50-HA) or 15% (RASCAL_TB40/E) SDS-PAGE gels. An aliquot corresponding to 2% of the original co-IP buffer volume was loaded onto the gels as the input control (input). Membranes were probed with anti-HA (1:500) or anti-RASCAL (1:1,000) Abs. (B) Immunoblot analysis of protein extracts from HEK293T cells transiently transfected with LNCX or cotransfected with LNCX UL50-HA and L-UL53-FLAG. Proteins were denatured in 3% SDS lysis buffer (lysate) or were subjected to co-IP with anti-HA Abs, prior to separation on 10% SDS-PAGE gels. Membranes were probed with anti-HA (1:500) or anti-FLAG (1:500) Abs. Expected molecular masses were 43.9 kDa for UL50-HA, 10.6 kDa for RASCAL_TB40/E, and 43.3 kDa for UL53-FLAG.