The X Protein of Borna Disease Virus Serves Essential Functions in the Viral Multiplication Cycle

Abstract

The X gene of Borna disease virus (BDV) encodes a nonstructural 10-kDa protein that can interact with viral polymerase cofactor P, thus regulating polymerase activity. It remained unknown whether X is essential for virus multiplication. All our attempts to generate mutant BDV with a nonfunctional X gene proved unsuccessful. However, a mutant virus with an inactive X gene was able to replicate in Vero cells if an artificial gene cassette encoding X was inserted at a site near the 5 end of the viral genome. These results indicate that X performs essential viral functions.The genome of Borna disease virus (BDV) codes for at least six proteins, of which five are present in viral particles (3,10,11). The sixth protein, denominated X or p10, is a 10-kDa nonstructural polypeptide (15) that associates with the viral polymerase complex in infected cells (17) but is not required for the formation of an active polymerase complex in artificial minireplicon systems (7, 13). X can specifically bind to viral polymerase cofactor P with the help of an interaction domain located near its N terminus (18). Wild-type X, but not X mutants lacking a functional P interaction domain, inhibits polymerase activity in artificial minireplicon systems (8), indicating that X is a polymerase regulator. It remained unclear, however, whether X is essential for virus multiplication.To address this question, we introduced specific mutations into the X gene of a full-length cDNA clone that was previously shown to yield infectious BDV upon transfection into suitable cells (6,14). Since the X and P proteins are translated from overlapping reading frames of a single mRNA, analysis of X protein function is difficult, as mutations in X might influence the expression of P. In construct i, we intended to specifically inactivate the P interaction domain in X by mutating the amino acids at positions 10 and 11 (Fig. 1). Previous studies with a viral minireplicon system showed that mutant XA10A11 lost the ability to inhibit BDV polymerase activity (8). In construct ii, we inactivated the X initiation codon by changing AUG to GCG and blocked translation of the X open reading frame by further introducing termination codons at amino acid positions 11 and 12 (Fig. 1). At least five attempts were made to generate infectious virus from each of these two constructs. None of the attempts was successful (Fig. 1). The deleterious effect of X gene inactivation was not overcome if we used rescue constructs with a modified L polymerase gene (LRD) (Fig. 1, constructs iv and v), which strongly improves the multiplication of an attenuated virus expressing green fluorescent protein (GFP) (12). Appropriate wild-type constructs that were used in parallel yielded virus in each trial (data not shown), excluding trivial technical problems.These results indicated that X serves essential functions and is required for virus multiplication in Vero cells. Since this preliminary conclusion was based on negative results, it remained possible that our failure to recover virus from the mut...

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