The Critical Role Of K1685 And K1829 In The Large Protein Of Rabies Virus On Viral Pathogenicity And Immune Evasion

Rabies, caused by rabies virus(RABV), is one of the oldest infectious zoonosis. There are around 55000 people die of rabies all over the world each year, most of which are from Asia and Africa. China is one of the countries with high incidence of rabies.RABV’s genome is a negative-strand RNA encoding five structural proteins: nucleoprotein(N), phosphoprotein(P), matrix protein(M), glycoprotein(G) and the large protein(L). Currently, there are very few studies related with L protein. Based on the studies of other nonsegmented negative-sense(NNS) RNA viruses, especially Ves icular Stomatitis Virus(VS V) that belongs to Rabdovir idae as RABV, there are conserved tetrad within the L protein related to 2’-O and N-7 MTase activities during viral m RNA capping process. These conserved tetrad play important roles in viral pathogenicity and innate immune evasion.In the present study, the amino acid sequence alignments were carried out through online software CLUSTAL W2 among the Carboxy-terminal of the L protein from VSV, measles virus, Sendai virus, Newcastle virus, and Nipha virus. The result indicated that RABV L protein exs its the K-D-K- E tetrad and the key amino acids that K, D, K, and E are very conserved. Moreover, the K-D-K-E tetrad was found to be conserved among different strains of RABV.To investigate the role of this K-D-K-E tetrad on RABV pahtogenicity and immue evasion, a serial of recombinant RABVs(r RABVs) with amino acid substitutions at the four key amino acids(1685K, 1797 D, 1829 K, and 1867E) in RABV L were constructed through reverse genetics system, and the pathogenicities of these recombinant RABV and parent virus were compared in vitro and in vivo. It was found that r B2c-D1797 A and r B2c-E1867 A reverted to the parent virus following the first two passages in BSR cells. While the other two mutants, rB2c-K1685 A and r B2c-K1829 A, were stable during at least the first 15 passages in cell culture. Therefore, r B2c-K1685 A and r B2c-K1829 A were chosen for further study. The virus titer and cellular spread ablity of these two mutants are lower than those of the parent virus. Furthermore, the mature mice infected with r B2c-K1685 A or r B2c-K1829 A did not show any clinical signs of rabies, and the suckling mice infected with r B2c-K1685 A or r B2c-K1829 A exhibited highly attenuated phenotype on body weight loss, clinical scores, and survivorship, compared with parent virus r B2 c.To further investigate whether the nutations at the K-D-K-E tetrad can also affect the immne evasion of RABV, the comparisons of produc tion of type-I IFN and the sensitivity to IFN were conducted between the mutant and parent virus. The results suggested that the mutants did not enhance the production of type-I IFN in RAW264.7 cells, but were more sensitive to the type I IFN compared with parent virus. Previous studies have shown that the interferon stimulated gene IFIT1/2 can specifically inhibit virus replication by recognizing viral m RNA lacking 2’-O methylation. Therefore, the IFIT1 or IFIT2 over-expression cells lines were constructed to investigate whether these ISGs can also affect RABV replication. The result indicated that the replication of r B2c-K1685 A and r B2c-K1829 A could be inhibited by IFIT2 but not IFIT1.Furthermore, the mice were immunized with the two mutants or parent vir us to investigate whether the immunogenicity was also affected by the mutations at the K-D-K-E tetrad The virus neutralizing antibody( VNA) titers were measured at two weeks post immunization, and the VNA levels were higher than 10 IU/ml in all the immuniz ed mice. The challenge exper iment was carried out at 3 weeks post immunization with CVS-24. All the mice immunized with r B2c-K1685 A or r B2 c were protected, and 90% of the mice immunized with r B2c-K1829 A were protected. Together, the mutation of K-D-K-E tetrad did not signif icantly affect the immunogenicity of RABV.Taken together, the mutations at K-D-K-E tetrad can attenuated the RABV to make it more sensitive to type-I IFN and IFIT2, but did not change the immunogenicity signif icantly. This study will not only suggest that K-D-K-E tetrad in the L protein play an important role in the pathogenicity and immune evasion during RABV infection but also facilitate the development of new live attenuated vaccines for RABV by inhibiting viral mRNA cap methylation.