Extreme dependence of gH and gL expression on ORF57 and association with highly unusual codon usage in rhesus monkey rhadinovirus

Abstract

Standard vectors for high-level expression elicited undetectable levels of the gH and gL glycoproteins of rhesus monkey rhadinovirus (RRV) following transient-transfection assays under a variety of conditions. These same vectors and conditions yielded high levels of RRV gB expression. Unlike other genes of RRV, both the gH and gL genes were noted to have a highly aberrant, suboptimal codon usage. High levels of RRV gH and gL expression were achieved by two alternative means: codon optimization or coexpression of RRV ORF57. The failure of gH and gL to be expressed in the absence of ORF57 and in the absence of codon optimization could not be explained by the failure of RNA to egress from the nucleus. Rather, the defect in gH and gL expression appeared to be cytoplasmic in nature. It is not clear at the present time whether the aberrant codon usage for gH and gL of RRV is an intentional regulatory strategy used by the virus or whether it is driven by some external force, such as intrinsic immunity. In any event, our results indicate that the need of ORF57 for gH and gL expression can be circumvented by codon optimization, that RRV ORF57 acts principally to allow translation of gH and gL RNA in the cytoplasm, and that this activity of ORF57 is related in some way to the aberrant codon usage of the gH and gL RNAs.

FIG. 1.

Achievement of gH and gL expression by codon optimization or by coexpression of ORF57. RRV gH, gL, and gB gene coding regions were cloned in-frame into the pEF1-myc/HisA expression vector (Invitrogen, Carlsbad, CA) after PCR amplification from the RRV26-95 cosmid library (1). RRV ORF57 was PCR amplified from the RRV26-95 cosmid library and inserted in-frame into the pEF1-V5/His expression vector (Invitrogen). Codon-optimized versions of RRV gH and gHt were PCR amplified from a template plasmid acquired through DNA2.0 (Menlo Park, CA) and inserted into the pEF1-myc/HisA expression vector. A codon-optimized version of RRV gL was PCR amplified from a template plasmid acquired through DNA2.0 and inserted into the pEF1-V5/HisA expression vector. Recloned products generated by PCR were sequenced to verify the absence of introduced mutations. One day postseeding, HEK293T (A to C) or Vero cells (D) (4.5 × 10⁵ cells/well in 6-well plates) were transfected with different combinations of expression plasmids using the Transfectin reagent (Bio-Rad Laboratories, Hercules, CA) using a scaled-down procedure. At 48 h posttransfection, cultures were rinsed with phosphate-buffered saline and lysed with radioimmunoprecipitation assay buffer (Boston BioProducts, Inc., Worcester, MA). Lysates were sonicated at 20% for 10 s with a Fisher Scientific sonic dismembrator (model 500), and debris was spun down at 14,000 × g in a microcentrifuge for 1 min. Protein concentrations were determined by bicinchoninic acid assay, following the manufacturer's instructions (Pierce Biotechnology, Rockford, IL). Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked overnight at room temperature in 5% milk in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) (Sigma, St. Louis, MO). Membranes were incubated for 1 h with mouse monoclonal antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 5% milk-PBS-T. The antibodies were specific for either Myc (for detection of gB, gH, c.o. gH, and gL in panels A, B, and D), six-His (for detection of gL and c.o. gL in panel C), or β-tubulin or V5 (for detection of ORF57 in all blots and c.o. gL in panel D). After successive washings in PBS-T, blots were incubated in secondary antimouse (Santa Cruz) diluted in 5% milk in PBS-T. Blots were washed in PBS-T, and antibody binding was detected using the SuperSignal West Pico chemiluminescent reagent (Pierce) and a Fuji phosphorimager. ORF57 is observed as a doublet because the transcript has multiple start sites for translation. Incubation of replicate blots or the same blot (for gB) with an anti-β-tubulin antibody was used to show equal loading. Mobility of the codon-optimized gL-V5-His protein differs slightly from that of the gL-myc-His protein because of the different epitope tag. Samples transfected with gH, c.o. gH, gL, or c.o. gL are indicated by brackets.

FIG. 2.

Gribskov codon usage charts. The codon usage profile for each gene was determined using MacVector with codon preferences for Homo sapiens.

FIG. 3.

Expression of other RRV genes in the presence and absence of ORF57. RRV genes for gHt (A), gM (B), gN (C), R8.1 (D), ORF56, ORF60, and ORF70 (E) were cloned into the pEF1-myc/HisA vector with a Myc/His epitope at the C terminus, expressed, and detected as described in the legend to Fig. 1.

FIG. 4.

Cytoplasmic and nuclear RNA after transfection of HEK293T cells. (A) gH. (B) gL. At 48 h posttransfection, total cytoplasmic RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA), following the instructions for cytoplasmic RNA purification. Total nuclear RNA was isolated from the nuclear pellets remaining from the cytoplasmic RNA isolation procedure. The concentration of purified cytoplasmic and nuclear RNA was determined by spectrophotometry. Five micrograms of RNA was combined with formamide loading buffer (Ambion, Austin, TX), denatured at 65°C for 10 min, and loaded onto a 1% denaturing formaldehyde agarose gel containing ethidium bromide in accordance with the instructions of the NorthernMax instruction manual (Ambion). The RNA was transferred to a BrightStar-Plus membrane (Ambion) overnight and then cross-linked by UV light exposure in a commercial cross-linker (Bio-Rad). Blots were then prehybridized in UltraHyb (Ambion) for >1 h at 42°C. An end-labeled antisense oligonucleotide probe was added directly to UltraHyb and allowed to hybridize overnight at 42°C. Antisense probes were generated by 2 h of incubation of [γ-³²P]ATP, antisense oligonucleotides (c-myc, 5′-CAGATCCTCTTCTGAGATGAGTTTTTGTTC-3′, or V5, 5′-ACCGAGGAGAGGGTTAGGGATAGGCTTACC-3′), and polynucleotide kinase (New England Biolabs, Beverly, MA). Following this incubation, the end-labeled probes were purified using ProbeQuant G-50 Micro columns (GE Healthcare, Pittsburgh, PA). The next day, the blots were washed in triplicate with low-stringency wash solution (Ambion) for 5 min at room temperature, followed by one wash in low-stringency wash solution for 10 min at 42°C. Blots were then exposed overnight in phosphorimager cassettes, and the screens were read using a phosphorimager (BAS 2000; Fuji Photo Film Co., Tokyo, Japan). Blots were then stripped in 0.1% sodium dodecyl sulfate, prehybridized as before, and hybridized to an antisense probe to Macaca mulatta cyclophilin A (5′-CCAAATCCTTTCTCTCCAGTGCTCAGAGC-3′) prepared as described above. Blots were rinsed, exposed to phosphorimager screens, and read as above. The lower panel for each shows the ethidium bromide-stained agarose gel. C, cytoplasmic; N, nuclear. After hybridization and exposure for detection of gH (A) or gL (B) RNA, the blot was stripped and reprobed for cyclophilin A as a control for equal RNA loading. Samples transfected with pUC19 (Invitrogen), gH, c.o. gH, gL, or c.o. gL are indicated by brackets.