Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs

Abstract

The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.

FIG 1

Size distribution of small RNAs expressed in DENV- and WNV-infected Huh7 cells. Human Huh7 cells were infected at an MOI of 1 with either DENV or WNV. Total RNA was harvested at 96 h postinfection for DENV or at both 48 h and 72 h postinfection for WNV. Small RNAs (17 to 29 nt in length) were then subjected to deep sequencing and aligned to the human or relevant viral genome. (A) Size distribution of small RNAs of human origin. (B) Size distribution of small RNAs derived from DENV. (C) Size distribution of small RNAs of WNV origin at 48 h and 72 h postinfection.

FIG 2

Genomic origin of small viral RNAs recovered in DENV- or WNV-infected Huh7 cells. Small viral RNAs recovered by deep sequencing, as described for Fig. 1, were aligned to their genomic location of origin on the DENV genome (A) or the WNV genome (B and C), with RNAs derived from the positive strand shown above the x axis and those originating from the viral negative strand below the x axis. As may be observed, almost all viral reads derived from the viral positive strand.

FIG 3

Analysis of the replication rates of three flavivirus species in cells lacking Dicer function. Either wild-type 293T cells or their Dicer-deficient clonal derivatives, NoDice(2-20) and NoDice(4-25) cells, were infected with DENV (A), WNV (B), or YFV (C) at an MOI of 1 (DENV and YFV) or 0.1 (WNV), and the levels of viral progeny released into the supernatant media were determined by measuring viral focus forming units (FFU) at the indicated time points. Averages of three replicates with standard deviations (SDs) are indicated.

FIG 4

Analysis of the replication rate of SINV, VEEV, or MeV in the presence or absence of Dicer function. Wild-type 293T cells or their Dicer-deficient derivatives, NoDice(2-20) and NoDice(4-25) cells, were infected with SINV at an MOI of 5 (A), VEEV at an MOI of 5 (B), or MeV at an MOI of 1 (C). Supernatant media were collected at the indicated times and assayed for infectious virus by measurement of PFU on BHK-21 (A and B) or Vero/hSLAM (C) cells. Data from representative duplicate experiments are presented as averages of triplicate samples with SDs indicated.

FIG 5

Analysis of the levels of IAV RNA expression in infected cells in the presence or absence of Dicer function. Wild-type 293T cells or their Dicer-deficient derivatives, NoDice(2-20) and NoDice(4-25) cells, were infected with IAV strain WSN at an MOI of 0.1, and viral replication was then assayed at the indicated times postinfection by quantitation of the level of the viral nucleoprotein genome segment by qRT-PCR. Cellular GAPDH mRNA was used as an internal control. Averages of three independent experiments with SDs are indicated.

FIG 6

Quantification of the level of reovirus replication in the presence or absence of Dicer function. Wild-type 293T cells or their derivative, NoDice(2-20) cells, were infected with reovirus strain T1L or T3D at an MOI of 3, and viral replication was then assayed at 22 h postinfection by plaque assay on mouse L929 cells. Averages of two wells with SDs are indicated. While results suggest a slightly lower level of replication in NoDice cells, NoDice cells replicate more slowly than the parental 293T cells and were less abundant at the time of harvest (data not shown), likely accounting for the minimal difference in titers.

FIG 7

Quantification of the levels of VSV replication and RNA expression in infected cells in the presence or absence of Dicer. Either wild-type 293T cells or their derivatives, NoDice(2-20) and NoDice(4-25) cells, were infected with a VSV derivative engineered to express GFP. (A) At 9 h postinfection, the supernatant media were harvested and viral progeny production was analyzed on BHK-21 cells by FACS analysis of the level of GFP-positive cells. (B) In parallel, total RNA was harvested from the transfected cells, and levels of VSV genomic RNA were quantified by qRT-PCR. Cellular GAPDH mRNA was used as an internal control. Averages of three independent experiments with SDs are indicated.

FIG 8

Analysis of HIV-1 infection and progeny production in the presence and absence of Dicer function. (A) Wild-type 293T cells or the NoDice(2-20) and NoDice(4-25) cell lines were infected with an equal amount of a previously described HIV-1 derivative engineered to express FLuc (56). At 24 h postinfection, the cells were lysed and the level of FLuc expression was quantitated. (B) Shown are the results of an experiment similar to that for panel A, except that the cells were infected with an HIV-1 derivative engineered to express GFP. The level of transduced cells was quantitated by FACS at 48 h postinfection. (C) 293T or NoDice cells were transfected with a plasmid encoding a full-length replication-competent HIV-1 provirus together with an internal control plasmid expressing GST. At 48 h posttransfection, the supernatant media and transfected cells were harvested, and the former was filtered and then used to infect the indicator cell line TZM-bl, which expresses FLuc only after HIV-1 infection. Induced FLuc expression levels were determined at 24 h postinfection. In parallel, the level of expression of the GST internal control in the transfected cells was determined by Western blotting and used to correct the level of FLuc expression (54). Averages of three independent experiments are shown in each panel with SDs indicated.

FIG 9

Analysis of HSV-1 infection in the presence and absence of Dicer function. Wild-type 293T cells or the NoDice(2-20) and NoDice(4-25) cell lines were infected with HSV-1 at an MOI of 0.1. Total DNA was harvested at 18 h postinfection, and HSV-1 genomic DNA was quantitated by qPCR. The human 18S rRNA gene served as an internal control. Averages of three independent experiments with SDs are indicated.