Tick-borne Nyamanini virus replicates in the nucleus and exhibits unusual genome and matrix protein properties

Abstract

Tick-borne Nyamanini virus (NYMV) is the prototypic member of a recently discovered genus in the order Mononegavirales, designated Nyavirus. The NYMV genome codes for six distinct genes. Sequence similarity and structural properties suggest that genes 1, 5, and 6 encode the nucleoprotein (N), the glycoprotein (G), and the viral polymerase (L), respectively. The function of the other viral genes has been unknown to date. We found that the third NYMV gene codes for a protein which, when coexpressed with N and L, can reconstitute viral polymerase activity, suggesting that it represents a polymerase cofactor. The second viral gene codes for a small protein that inhibits viral polymerase activity and further strongly enhances the formation of virus-like particles when coexpressed with gene 4 and the viral glycoprotein G. This suggests that two distinct proteins serve a matrix protein function in NYMV as previously described for members of the family Filoviridae. We further found that NYMV replicates in the nucleus of infected cells like members of the family Bornaviridae. NYMV is a poor inducer of beta interferon, presumably because the viral genome is 5' monophosphorylated and has a protruding 3' terminus as observed for bornaviruses. Taken together, our results demonstrate that NYMV possesses biological properties previously regarded as typical for filoviruses and bornaviruses, respectively.

Fig 1

NYMV replicates in the nucleus. (A) Specificity for NYMV antigens of serum from NYMV-infected mice. 293T cells were transfected with expression vectors encoding the indicated NYMV proteins or the empty vector as a control (Ø). At 24 h posttransfection the cells were lysed, and Western blot analysis was performed. Antibodies against β-tubulin were used to control for equal loading of the gel. (B) Immunofluorescence analysis. Infected (NYMV) and uninfected (Ø) Vero cells were stained with DAPI and NYMV antiserum. (C) Subcellular distribution of NYMV proteins. Whole-cell lysates as well as cytoplasmic (Cy) and nuclear (Nu) fractions were prepared from infected and uninfected Vero cells. Samples were subjected to immunoblotting using primary antibodies against histone H3 or β-tubulin and NYMV antiserum. (D) Subcellular distribution of viral RNA. Samples of RNA extracted from whole-cell lysates or cytoplasmic and nuclear fractions were subjected to Northern blot analysis using a DNA probe corresponding to nucleotides 138 to 1971 of the NYMV antigenome, which detects RNAs containing N and ORF2. n.i., not infected.

Fig 2

NYMV multiplication is dependent on cellular RNA polymerase II activity. Vero cells were infected with NYMV, VSV, or WSN using an MOI of 1. Immediately after infection, the cells were either treated with 5 μg/ml of actinomycin D (gray bars) or left untreated (black bars). Viral titers were determined at 24 h (for VSV) or at 40 h (for NYMV and WSN) postinfection by the 50% tissue culture infective dose (TCID₅₀) assay. Bars represent the average values of two independent experiments. Standard deviations are shown. Significance was tested using the two-tailed t test.

Fig 3

ORF3 of NYMV codes for a polymerase cofactor. (A) Schematic representation of the NYMV genome. Transcriptional start (S1–S6) and stop (T1–T6) signals are indicated. Protein-coding regions are shown as gray boxes. (B) Reconstitution of the NYMV polymerase complex. BSR-T7 cells were transfected with the NYMV minigenome encoding Gaussia luciferase, expression vectors encoding N and L, and plasmids encoding the indicated additional NYMV proteins. To normalize for transfection efficacy, a plasmid encoding firefly luciferase was cotransfected. At 72 h posttransfection, Gaussia and firefly luciferase-mediated light emissions were quantified. The normalized Gaussia luciferase RLU value obtained for the cells transfected with expression vectors for N, ORF3, and L was arbitrarily set to 100%. Bars show the average values of two independent duplicate experiments. Standard deviations are shown. (C) Interaction of the ORF3 product with other NYMV proteins in mammalian two-hybrid assays. 293T cells were transfected with a Gal4 promoter-dependent firefly luciferase construct, pCA-VP16/ORF3, and the indicated pCA-Gal4 constructs. A constitutive active Renilla luciferase reporter construct was cotransfected for normalization. At 24 h posttransfection, Renilla and firefly luciferase-mediated light emissions were quantified. BDV-P fused to the Gal4-binding domain served as the negative control; its normalized firefly RLU value was arbitrarily set to 1. Bars show the average values of three independent experiments. Standard deviations are shown. RLU = relative light units.

Fig 4

ORF2 negatively regulates NYMV polymerase activity. (A) BSR-T7 cells were transfected with the NYMV minigenome encoding Gaussia luciferase and the indicated pCA expression vectors. To normalize for transfection efficiency, a plasmid encoding firefly luciferase was cotransfected. At 72 h posttransfection, Gaussia and firefly luciferase-mediated light emissions were quantified. The normalized value obtained for the positive control (containing pCA-N, -ORF3, and -L only) was arbitrarily set to 1. Bars represent the average values of three independent experiments. Standard deviations are shown. (B) The inhibitory effect of ORF2 on viral polymerase activity is dose dependent. BSR-T7 cells were transfected as in panel A but with increasing amounts of the ORF2-encoding plasmid. The analysis was performed as described for panel A. The normalized value obtained for the positive control (containing pCA-N, -ORF3, and -L only) was arbitrarily set to 100%. Bars show the average values of three independent experiments. Standard deviations are shown. RLU = relative light units.

Fig 5

Proteins encoded by ORF2 and ORF4 cooperate for the production of infectious VLPs. BSR-T7 cells were transfected with the NYMV minigenome encoding Gaussia luciferase and the indicated pCA expression vectors. Supernatants were collected at 72 h posttransfection and clarified through a 0.45-μm filter. Five hundred microliters of the clarified supernatants were transferred onto 293T cells previously transfected with pCA-N, pCA-ORF3, and pCA-L. Forty-eight hours later, the 293T cells were lysed and Gaussia-mediated light emission was quantified. The value obtained for the negative control (receiving only pCA-N, -ORF3, and -L) (lane 1) was arbitrarily set to 1. Bars represent the average values of three independent experiments. Standard deviations are shown. RLU = relative light units.

Fig 6

Products of ORF2 and ORF4 interact with each other but not with G. 293T cells were transfected with pCA expression vectors encoding HA-tagged ORF2 together with Flag-tagged ORF4 or Flag-tagged G (A) or HA-tagged ORF4 together with Flag-tagged ORF2 or Flag-tagged G (B). At 24 h posttransfection, protein extracts were subjected to immunoprecipitation using anti-Flag M2 beads. Flag- and HA-tagged proteins were detected using specific antibodies. (C) The ORF2-ORF4 protein complex does not interact with G. 293T cells were transfected with pCA expression vectors encoding HA-tagged ORF2, Flag-tagged G, and untagged ORF4. Immunoprecipitation was performed as described for panels A and B. The two bands detected for G/Flag presumably represent immature and cleaved forms of G. IP = immunoprecipitation; IB = immunoblotting.

Fig 7

NYMV RNA is a poor inducer of IFN-β. Five hundred nanograms of RNA isolated from purified viral particles (A) or infected cells (B) was transfected into 293T cells that were transfected with a plasmid coding for firefly luciferase under the control of the IFN-β promoter. Where indicated, the viral RNA was treated with shrimp alkaline phosphatase (SAP) prior to transfection. Firefly luciferase activity was normalized for transfection efficacy, and the value for the mock control was arbitrarily set to 1. Bars represent the average values of three independent experiments. Standard deviations are shown. RLU = relative light units.

Fig 8

The NYMV genome is monophosphorylated and contains noncomplementary nucleotides at the 3′ end. (A) Analysis of terminal genome sequences by RACE. For 5′ RACE, a synthetic RNA (oligonucleotide) was ligated to viral RNA (vRNA) without prior pyrophosphatase treatment. For 3′ RACE, viral RNA was terminally elongated with poly(A) (right panel) or poly(C) (middle panel). Electropherograms from direct sequencing of PCR products are shown. (B) Deduced structure of the NYMV genome in the panhandle conformation. The molecule is predicted to have a monophosphate at the 5′ end and a 3′ overhang (gray box).