Selective uptake of small RNA molecules in the virion of murine gammaherpesvirus 68

Abstract

Noncoding RNAs are a feature of many herpesvirus genomes. They include microRNAs, whose function is the subject of intense investigation, in addition to longer RNA molecules such as the Epstein-Barr virus-encoded RNAs and herpesvirus saimiri U RNAs, which have been known for some time but whose function is still not well defined. Murine gammaherpesvirus 68 (MHV-68) encodes eight viral tRNA-like molecules (vtRNAs) of unknown function. Investigating the kinetics of expression of the vtRNAs, we observed that they were present directly after infection with the virus. This strongly suggested that vtRNAs were part of the virion structure, which was confirmed by their detection within various purified, RNase-treated preparations. Although both viral and cellular mRNAs were also detected within the MHV-68 virion, the major RNA species present were small RNAs of around 70 nucleotides in length. Interestingly, incorporation of viral mRNA was not related to the relative abundance in infected cells, as M11 mRNA, which is present at low abundance, was found in virions. MHV-76, which lacks the genes encoding the vtRNAs, also incorporated small RNA molecules within the virion, suggesting a requirement for these molecules for virion maturation. In productively infected cells the vtRNAs localized predominantly within the cytoplasm, although they also exhibited a globular pattern of nuclear staining. Their presence in the cytoplasm is consistent with interaction with virion components prior to maturation of virus particles. The significance of these findings for virion architecture and function is discussed.

FIG. 1.

(A) Expression of vtRNA1 in C127 cells infected with MHV-68. C127 cells were infected at an MOI of 5 PFU/cell, and at various times, the RNA was harvested and subjected to RT-PCR for vtRNA1. Experiments were carried out in the presence (+RT) or absence (−RT) of reverse transcriptase. (B) RNA was isolated from the virus stock and from infected cells at 0 hpi and subjected to RT-PCR for vtRNA1.

FIG. 2.

vtRNAs are present in purified MHV-68 virions. (A and B) Cell-associated MHV-68 virions were purified through a sucrose cushion, and RNA was extracted and subjected to RT-PCR. (A) RNA was subjected to RT-PCR for vtRNA1 or treated with RNase One followed by RT-PCR, showing that the vtRNA1 product was derived from RNA. (B) RT-PCR of virion RNA showing the presence of vtRNA2 to -8 and mRNAs for β-actin, GAPDH, and M3 in virion preparations. (C) RT-PCR on RNA extracted from MHV-68 grown in BHK-21 cells and purified through a sucrose gradient. RT-PCR was carried out for vtRNA1 and β-actin on virus taken from the upper band, lower band, and virus band. Experiments were carried out in the presence (+) and absence (−) of reverse transcriptase. Positive (+ve) and negative (−ve) controls were included in the reactions.

FIG. 3.

Analysis of the RNA species present within purified virus stocks. MHV-68 and MHV-76 were grown in mouse embryonic fibroblast cells lacking the type I IFN receptor and purified from cell-free supernatants by centrifugation though a sucrose cushion. Virions were treated with RNAse One prior to extraction of RNA. RNA was labeled with [³²P]CTP and fractionated on a 3% agarose gel (A), a 10% polyacrylamide-urea gel together with a mock preparation (B), and a Northern blot of purified virion RNAs probed with [³²P]CTP-labeled RNA probe specific for the first 20 nucleotides of vtRNA1 (C). Numbers at left of each panel are molecular sizes in nucleotides.

FIG. 4.

In situ hybridization for vtRNA1 to -4. C127 cells were infected at an MOI of 5 PFU/cell for 24 h and probed with a digoxigenin-labeled RNA probe specific for vtRNA1 to -4. Detection of the digoxigenin-labeled probe was carried out using biotinylated antidigoxigenin and streptavidin-Alexa Fluor 488. Sections were counterstained with propidium iodide (PI) and imaged using a Leica TCS-NT confocal microscope. Treatment with RNase was also carried out prior to hybridization.