The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles

The vaccinia virus (VV) A10L gene codes for a major core protein, P-la, Thi s polypeptide is synthesized at late times during viral infection and is pr oteolytically cleaved during virion assembly. To investigate the role of P4 a in the virus life cycle and morphogenesis, we have generated an inducer-d ependent conditional mutant (VVindA10L) in which expression of the A10L gen e is under the control of the Escherichia coli lad operator/repressor syste m. Repression of the A10L gene severely impairs virus growth, as observed b y both the inability of the virus to form plaques and the 2-log reduction o f viral yields. This defect can be partially overcome by addition of the in ducer isopropyl-beta -D-thiogalactopyranoside (IPTG). Synthesis of viral pr oteins other than Pla occurred, although early shutoff of host protein synt hesis and expression of viral late polypeptides are clearly delayed, both i n the absence and in the presence of IPTG, compared with cells infected wit h the parental virus. Viral DNA replication and concatemer resolution appea red to proceed normally in the absence of the A10L gene product. In cells i nfected with WindA10L in the absence of the inducer virion assembly is bloc ked, as defined by electron microscopy, Numerous spherical immature viral p articles that appear devoid of dense viroplasmic material together,vith hig hly electron-dense regular structures are abundant in VVindA10L-infected ce lls. These regularly spaced structures can be specifically labeled with ant i-DNA antibodies as well as with a DNase-gold conjugate, indicating that th ey contain DNA, Some images suggest that these DNA structures enter into sp herical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after form ation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.