Expression and mutational analysis of Autographa californica nucleopolyhedrovirus HCF-1: functional requirements for cysteine residues.
The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.
Wilson
Joyce A
JA
Departments of Entomology, University of Georgia, Athens, Georgia 30602, USA. jwilson@uhnres.utoronto.ca
Forney
Scott D
SD
Ricci
Alessondra M
AM
Allen
Emily G
EG
Hefferon
Kathleen L
KL
Miller
Lois K
LK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D016384:Consensus Sequence; D003545:Cysteine; D017931:DNA Primers; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; C507354:HCF-1 protein, Autographa californica nucleopolyhedrovirus; D014764:Viral Proteins; D003545:Cysteine