RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration

Abstract

The assembly of human cytomegalovirus (HCMV) virions is a complex process and involves the incorporation of viral transcripts. These RNAs are delivered to the newly infected cells and have the potential to be translated in the absence of HCMV gene expression. We have quantified the relative amount of RNAs in HCMV virions and infected cells with real-time reverse transcription-PCR and observed that viral and cellular RNAs are packaged in proportion to the amount of RNA within the cell at the time of assembly. To determine whether cis elements influenced RNA packaging, we constructed a recombinant HCMV mutant virus that expressed the yellow fluorescence protein (YFP) gene fused to the virion RNA UL21.5. We also constructed a mutant virus in which the UL21.5 transcription unit was replaced with the YFP gene. YFP RNA was incorporated into both viruses, indicating that RNA is incorporated in the absence of a virus-specific signal motif. Furthermore, with in situ hybridization, packaged transcripts were observed throughout the cytoplasm of the infected cells, including the site of virus assembly. Several proteins that nonspecifically interact with RNA, including the tegument protein pp28, were found within HCMV virions. These studies demonstrate that both viral and cellular RNAs are nonspecifically incorporated into HCMV, potentially through interactions with several virion proteins.

FIG. 1.

Relative quantitation of RNA packaged into HCMV particles with real-time RT-PCR. (A) Real-time RT-PCR was completed with RNA isolated as described in Materials and Methods from fibroblasts and cell-free virus following 72 h of infection. cDNA was synthesized with random hexamers, and reactions were completed with primers to numerous viral and cellular genes with SYBR green detection. The results are presented as the ratio of the C_T value obtained from the virion RNA sample to the C_T value obtained from RNA isolated from infected cells. The ratios were normalized to the ratio for UL21.5 and are presented as the inverse value. An estimate of the packaging efficiency is shown when 10-fold more RNA is packaged as well as 0.1-fold less RNA relative to UL21.5. The data are presented as the standard deviation from two experiments. (B) Northern blot analysis was completed with an equal volume of virion RNA and 2 μg of total RNA from infected cells. Specific transcripts were detected with ³²P-labeled strand-specific probes to UL21.5, UL83, and UL107. Additional low-molecular-weight bands observed in virion RNA samples are indicated by lines. The locations of marker RNAs are indicated to the left of the gel, with sizes in kilobases.

FIG. 2.

Relative quantitation of RNA packaged into different HCMV particles types with real-time RT-PCR. RNA was isolated from each particle type and used to quantify the relative amounts of RNA packaged. The results are presented as the ratio of C_T values relative to the ratio for UL21.5 and are presented as the inverse value. The data are presented as the standard deviation from two experiments. NIEP, noninfectious enveloped particles; DB, dense bodies.

FIG. 3.

Quantitation with real-time RT-PCR of YFP-containing transcripts from recombinant HCMV viruses. (A) The recombinant virus BADsubUL21.5 was constructed by replacing the UL21.5 transcription unit with a cassette containing the kanamycin resistance (kan) and β-galactosidase (lacZ) genes. The cassette was replaced with the UL21.5 gene fused with the YFP gene in BADinUL21.5YFP and with the YFP gene and the polyadenylation signal (pA) from simian virus 40 virus in BADsubUL21.5YFP. (B) Total RNA was isolated from fibroblasts infected for 72 h and cell-free virus for both HCMV recombinant strains BADinUL21.5YFP and BADsubUL21.5YFP. Quantitative RT-PCR was completed with primers specific to RNAs containing YFP and T/IRL 7. The results are presented as the ratio of the C_T value obtained from the virion RNA sample to the C_T value obtained from RNA isolated from infected cells. The ratios were normalized to the C_T ratio for T/IRL 7 and are presented as the inverse value. The data are presented as the standard deviation from two experiments.

FIG. 4.

Localization of several packaged RNAs in infected fibroblasts during HCMV assembly. Fibroblasts were infected at a multiplicity of infection of 0.01 PFU/ml and processed for combined immunofluorescence and in situ hybridization at 72 h postinfection. In situ hybridization was completed with probes to UL21.5 (A to D), UL83 (E to H), and β-actin (I to L), shown in green. Immunofluorescence was completed with an antibody to the tegument protein pp28 (A, E, and I), shown in red. DNA was counterstained and appears blue (C, G, and K). Merged multicolor images (D, H, and L) and bars representing 10 μm (C, G, and K) are also shown.

FIG. 5.

Several proteins within HCMV particles interact nonspecifically with RNA. (A) Virion proteins isolated from cell-free virus were separated on a sodium dodecyl sulfate-10% polyacrylamide gel and transferred to nitrocellulose. Membranes were washed with decreasing amounts of guanidine HCl and incubated with ³²P-labeled UL21.5 (lane 2) and UL83 (lane 3) sense-strand RNA probes. Seven species consistently observed to bind radiolabeled RNA are indicated by arrows. Lane 1 shows total virion proteins stained with Ponceau S. The masses of marker proteins (in kilodaltons) are indicated to the left of the gel. (B) Electrophoretically separated proteins isolated from cell-free virus were hybridized to ³²P-labeled UL21.5 sense-strand RNA probes (lane 4) or an antibody to the tegument protein pp28 (lane 5). (C) Affinity purification with UL21.5 RNA bound to magnetic beads and proteins isolated from cell-free virus. In vitro-transcribed UL21.5 RNA was covalently attached to biotin and bound to streptavidin-coated magnetic beads. Proteins isolated from cell-free virus were incubated with bead-RNA complexes (lane 8) or beads alone (lane 7). Bound proteins were eluted and subjected to Western blot analysis with an antibody to pp28. Lane 6 represents crude virion extract.