Rescue of recombinant Marburg virus from cDNA is dependent on nucleocapsid protein VP30

Abstract

Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.

FIG. 1.

Cloning strategy for the full-length clone. (Left) Schematic drawing of the cassettes used for cloning. The numbers indicate nucleotide positions in the MARV genome (strain Musoke; GenBank accession number DQ217792). The MARV-specific sequence was obtained either by RT-PCR with viral RNA as the template or PCR assay of already existing plasmids derived from viral RNA. The SspI site shown in pMARV ApaI/SacI serves as a genetic marker. Restriction sites flanking the cassettes were used for construction of full-length pMARV(+) (right).

FIG. 2.

Characterization of recMARV. Virus was recovered from transfected BSR T7/5 cells mixed with Vero cells as described in the text. Vero cells in 25-cm² flasks were inoculated with the lysate of the mixed culture and checked for CPE daily. As a control, the plasmid encoding the L gene was omitted from the tranfection (−pT/L). (A) Light microscopic picture of CPE caused by wt MARV and recMARV on day 6 p.i. (B) Determination of viral titers. Vero cells in 96-well plates were infected with serial dilutions of the virus to determine the viral titer. After 12 days, the CPE was evaluated and the TCID₅₀ calculated. (C) Confirmation of the genetic marker. Virus from the supernatant of infected Vero cells was purified through a sucrose cushion at day 6 p.i. Viral RNA was isolated, reverse transcription was performed (except −RT, lane 5), and first-strand cDNA was subjected to PCR. The 632-bp product (lanes 1 and 3) was purified and digested with SspI where stated. Two bands at 301 and 331 bp, respectively, indicated the presence of the genetic tag in recMARV GP gene (lane 4). ctrl, control. (D) Immunofluorescence analysis of Vero cells infected with recMARV or wt MARV. Vero cells were grown on glass coverslips and infected with wt MARV or recMARV at a multiplicity of infection of 1. At 48 h p.i., virus was inactivated in 4% paraformaldehyde overnight. Immunofluorescence analysis was performed using a monoclonal mouse anti-MARV NP antibody and rhodamine-conjugated goat anti-mouse immunoglobulin G; nuclei were stained with DAPI. Arrows indicate viral inclusion bodies. (E) Electron microscopic pictures of purified virions. Virus was collected from the supernatant of infected Vero cells at 6 days p.i. and purified through a sucrose cushion. Virus was inactivated overnight in 4% paraformaldehyde and prepared for electron microscopy as previously described (26). Pictures were taken on a Zeiss 109 electron microscope at a magnification of ×50,000.

FIG. 3.

Localization and functionality of VP30_{Zn-finger knockout}. (A) Putative Zn-binding domain of MARV VP30. Two mutations (underlined) were introduced into pT/VP30_{M Zn-finger knockout} to disrupt the putative Zn-binding motif. The nucleotide numbers indicate positions in the MARV genome. (B) BSR T7/5 cells were grown on glass coverslips and transfected with pT/NP_M (NP), pT/VP30_M (VP30), and pT/VP30_{M Zn-finger knockout} (VP30_{Zn-finger ko}) as indicated on the left. At 48 h posttransfection, cells were subjected to immunofluorescence analysis using a monoclonal mouse anti-NP (α-NP) and a guinea pig anti-VP30 (α-VP30) antibody. NP was stained with rhodamine-conjugated goat anti-mouse antibodies and VP30 with fluorescein isothiocyanate-conjugated goat anti-guinea pig antibodies. The arrows indicate typical inclusion bodies formed by NP. (C) Western blot analysis of VP30_{M Zn-finger knockout}. BSR T7/5 cells were transfected with 1.0 and 2.0 μg, respectively, of either pT/VP30_M or pT/VP30_{M Zn-finger knockout} and analyzed by Western blotting at 2 days posttransfection. VP30_M was detected using a monoclonal mouse anti-VP30_M (α-VP30_M) antibody and a horseradish peroxidase-labeled goat anti-mouse antibody. (D) Transcriptional activity of VP30_{M Zn-finger knockout} using the EBOV-specific minigenome system. BSR T7/5 cells were transfected with plasmids encoding the nucleocapsid proteins of EBOV and the minigenome 3E-5E (27). EBOV VP30 was replaced with 2.0 μg of VP30_M or 2.0 μg of Zn-finger knockout (ko) mutants of the MARV-specific VP30_M (VP30_{M Zn-finger knockout}) or the EBOV-specific VP30_EBO (VP30_{E H90L}) (15). Cells were lysed after 48 h and subjected to a CAT assay. +, pT/L present; −, pT/L absent.

FIG. 4.

Influence of VP30 on recovery of recombinant MARV and EBOV. (A) BSR T7/5 cells were transfected with plasmids coding for MARV nucleocapsid proteins NP, L, and VP35, the full-length clone pMARV(+), and pC-T7Pol. A plasmid encoding either MARV VP30 (lane 1), the MARV VP30 Zn-finger mutant (VP30_{Zn-finger ko}, lanes 5 to 7), or EBOV VP30 (lanes 8 to 10) was added to the reaction mixture or VP30 was omitted from the transfection (w/o VP30, lanes 2 to 4). Except for the positive control (MARV VP30), transfection was performed in triplicate. At 6 days posttransfection, BSR T7/5 cells were mixed with Vero cells and lysed after an appropriate incubation period. Fresh Vero cells were infected with the supernatants and incubated until day 10 p.i. Cells were lysed, and total RNA was isolated and subjected to a one-step RT-PCR using primers targeting the MARV GP gene (nt 5890 to 6521). Ten percent of the reaction mixture was run on a 2% agarose gel and visualized with ethidium bromide. As controls, cells were infected with MARV (MARV wt inf., lane 12) or not infected (mock, lane 11). (B) BSR T7/5 cells were transfected with plasmids encoding EBOV NP, L, VP35, the full-length clone pFL-EBOVe+, and either VP30_EBO or the Zn-finger mutant VP30_{E H90L} (EBOV VP30_{Zn-finger ko}). At 6 days posttransfection, supernatants were used to infect Vero E6 cells. CPE caused by virus infection was determined after an incubation period of 6 days.