Attenuation of mouse hepatitis virus by deletion of the LLRKxGxKG region of Nsp1

Abstract

Coronaviruses are a family of large positive-sense RNA viruses that are responsible for a wide range of important veterinary and human diseases. Nsp1 has been shown to have an important role in the pathogenetic mechanisms of coronaviruses in vivo. To assess the function of a relatively conserved domain (LLRKxGxKG) of MHV nsp1, a mutant virus, MHV-nsp1-27D, with a 27 nts (LLRKxGxKG) deletion in nsp1, was constructed using a reverse genetic system with a vaccinia virus vector. The mutant virus had similar growth kinetics to MHV-A59 wild-type virus in 17CI-1 cells, but was highly attenuated in vivo. Moreover, the mutant virus completely protected C57BL/6 mice from a lethal MHV-A59 challenge. To further analyze the mechanism of the attenuation of the mutant virus, changes in reporter gene expression were measured in nsp1- or nsp1-27D-expressing cells; the results showed that nsp1 inhibited reporter gene expression controlled by different promoters, but that this inhibition was reduced for nsp1-27D. The research in vivo and in vitro suggests that the LLRKxGxKG region of nsp1 may play an important role in this process.

Figure 1. Sequence alignment of nsp1 from SARS-CoV and MHV-A59.

The conserved amino acids are indicated in the middle line. A region (LLRKxGxKG: amino acids 191–199) in nsp1 that is conserved in different coronaviruses, such as MHV and SARS-CoV, is shown by the blue box.

Figure 2. Organization of the MHV-A59 genome, and the relatively conserved region of nsp1 that was deleted in the mutated virus.

The genome is a 29.7-kb positive-sense RNA molecule that is capped (dark line) and polyadenylated (arrow). Genes are indicated for the replicases (ORF 1a and ORF 1b; white), structural proteins [Spike (S), Envelope (E), membrane (M) and nucleocapsid (N); black] and accessory proteins. ORF 1b is accessed by a ribosomal frameshift in the nsp12 coding sequence. The ORF 1a/b polyprotein is translated directly from input genome RNA, and processed into 16 mature nsps by two virus-encoded proteinases. Nsps have predicted or demonstrated activities as described in the text. The residues (27 nt) putatively involved in the formation of the conserved region of MHV-A59 nsp1 are shaded; the nsp1-27D was constructed by deletion of these conserved amino acids.

Figure 3. Sequence analysis and growth kinetics measurements of MHV-A59 and MHV-nsp1-27D.

(A) WT MHV-A59 sequence. (B) MHV-nsp1-27D sequence, with deletion of nts 780–807 of nsp1. (C) Comparison of MHV-A59 and MHV-nsp1-27D growth. 17Cl-1 cells were infected with MHV-A59 or MHV-nsp1-27D at an MOI of 1 pfu/cell. Samples of culture medium were obtained at 0, 2, 4, 6, 8, 10, 12 and 24 h p.i., and viral titers were determined by plaque assay. Each time point indicates the mean titer and standard deviation obtained from a triplicate series of infections.

Figure 4. MHV-nsp1-27D is highly attenuated in mice.

Groups of mice were infected intracranially (A, B) or intraperitoneally (C) with the indicated dose of MHV-nsp1-27D or MHV-A59. The survival rates of mice were monitored daily. For mice infected intraperitoneally with 5×10⁶ pfu of MHV-nsp1-27D or MHV-A59 (C), the animal weight was monitored daily. Each time point represents the mean data from at least five individual mice.

Figure 5. MHV-nsp1-27D is highly attenuated in vivo.

C57BL/6 mice were infected intraperitoneally with the indicated dose of MHV-nsp1-27D or MHV-A59. Serum ALT values (A, B, C, D) were measured at the indicated time points. Virus titers in the liver (E) and spleen (F) were determined at the indicated time points.

Figure 6. Hematoxylin and eosin staining of liver sections (40×).

A: Liver section from a normal mice. B: Liver section from a mouse of the MHV-WT group, on day 5 (intra-abdominal infection, 5×10³ pfu/animal). C: Liver section from a mouse of the MHV-nsp1-27D group, on day 8 (intra-abdominal infection, 5×10³ pfu/animal). D: Liver section from a mouse of the MHV-nsp1-27D group on day 15 (intra-abdominal infection, 5×10³ pfu/animal). The liver of the MHV-WT infected mice showed obvious lesions on day 5. The hepatocytes in the center of the lesion were fibroblast-like and without nuclei. In MHV-nsp1-27D infected mice, the hepatocytes showed unclear borderlines and increased gaps on day 8, which was restored to a normal state on day 15.

Figure 7. MHV-Nsp1-27D protects mice from challenge with WT MHV-A59.

Groups of C57BL/6 mice (n = 6) were immunized (A) intraperitoneally (5×10³ pfu/mouse) or (B) intracranially (200 pfu or 2×10⁴ pfu/mouse) with MHV-nsp1-27D, or treated with PBS (A). 14 days p.i., mice were challenged with 5×10⁶ (A) or 2×10⁴ (B) pfu/mouse of WT MHV-A59. The survival of mice was monitored daily.

Figure 8. MHV nsp1 and nsp1-27D reduce the expression of reporter genes controlled by different promoters.

BHK-21 cells were co-transfected with pRLuc-CMV (CMV promoter), pGL3-control (SV40 promoter) or pLuc-ISRE (ISRE promoter), and the indicated expression plasmids. 24 h post transfection, the luciferase activity was measured. The results represent the means of at least four independent experiments. Statistical analysis was performed using paired Student's t-test (* p<0.01).

Figure 9. IFN-ß levels in L929 cells expressing either nsp1 or nsp1-27D.

L929 cells were transiently transfected with the recombinant plasmids, pcDNA3.1-nsp1 or pcDNA3.1-nsp1 mu, and stimulated with NDV. The IFN-β level in the supernatant was determined using an ELISA kit. The IFN-β levels in the supernatant of cells transfected with pcDNA3.1-nsp1 and pcDNA3.1-nsp1 mu were decreased (130.8 pg/ml and 122.4 pg/ml) compared with cells transfected with control pcDNA3.1 (149.9 pg/ml). n.s., p>0.05.