The amino-terminal residue of Sindbis virus glycoprotein E2 influences virus maturation, specific infectivity for BHK cells, and virulence in mice

H W Heidner, R E Johnston
1994 Journal of Virology  
The E2 glycoprotein of Sindbis virus is synthesized as a precursor, PE2, which is cleaved by furin or a furin-like host cell protease at a late stage of maturation. The four-residue PE2 cleavage signal conforms to the basic amino acid-X-basic-basic motif which is present in many other viral and cellular glycoproteins which are processed by the cellular enzyme(s). In this report, we present evidence that the amino acid which immediately follows the signal, the N-terminal residue of E2, can
more » ... ue of E2, can influence protease recognition, binding, and/or cleavage of PE2. Constructs encoding nine different amino acids at E2 position 1 (E2 1) were produced by site-directed mutagenesis of the full-length cDNA clone of our laboratory strain of Sindbis virus AR339 (pTRSB). Viruses derived from clones encoding Arg (TRSB), Asp, Ser, Phe, His, and Asn in a nonglycosylated form at E2 1 contained predominantly E2. Viruses encoding Ile, Leu, or Val at E2 1 contained the uncleaved form of PE2. The specific infectivity of TRSB (E2 Arg-1) for baby hamster kidney (BHK-21) cells was from 5to greater than 100-fold higher than those of isogenic constructs with other residues at E2 1, suggesting that E2 Arg-1 represents a BHK-21 cell adaptive mutation in our laboratory strain. In newborn CD-1 mice, TRSB was more virulent than the PE2-containing viruses but less virulent than other PE2-cleaving viruses with alternative amino acids at E2 1. These results indicate that in TRSB, E2 Arg-1 increased the efficiency of virus-cell interactions in cultured BHK-21 cells but simultaneously decreased the ability of virus to mediate in vivo virus-cell interactions critical for the induction of disease. This suggests that the N terminus of E2 may participate in or be associated with virion domains which mediate these viral functions. Many viral glycoproteins are derived from larger precursors which are cleaved at specific sites during their maturation (63). Proteolytic processing can be required for the export of these proteins, for virus morphogenesis, and for the normal functional properties of the released virus particles (see reference 8 and references within for review). For many enveloped RNA viruses, including the alphaviruses (Togaviridae family), cleavage of the precursor is performed by a cellular protease(s) (24, 34, 61) . Viral substrates for the host cell protease(s) contain a highly conserved sequence motif which functions as the signal for site-specific cleavage. This signal is similar in its arrangement of basic amino acids to that found in specific membrane and secretory proteins which are the natural cellular substrates for the enzyme(s) (17). Alphaviruses provide a useful system for analyzing the sequence requirements of the cleavage signal itself, and for investigating how the cleavage event influences specific steps in the normal viral replication cycle (15, 29, 46) . Alphaviruses are small, relatively simple, enveloped viruses (49). The alphavirus genome consists of a positive-sense, single-stranded RNA which is encapsidated within a T=4 nucleocapsid composed of 240 copies of capsid (C) protein (2, 39). The nucleocapsid is contained within a host cell-derived lipid envelope (16). Two transmembranal, viral glycoproteins, El and E2, are exposed on the surface of the virion (44,48). El and E2 are associated as heterodimers, which in turn are organized in groups of three to form the viral spikes (44). The viral spikes form an icosahedral array at the virus surface and are arranged with T=4 symmetry (11, 38, 57) . The structural proteins of alphaviruses are synthesized as a * Corresponding author.
doi:10.1128/jvi.68.12.8064-8070.1994 fatcat:4msboel62nhdzat53bee6xvk5e