Expression and Mutational Analysis of Autographa californica Nucleopolyhedrovirus HCF-1: Functional Requirements for Cysteine Residues

Abstract

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.The family Baculovirudae is a diverse group of viruses that infect primarily insects. Baculoviruses are used routinely as expression vectors but are also interesting because of their potential use as biological insecticides (32). Most baculovirus species infect a narrow range of host insects. The type baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is unusual in that it has a host range that spans many insect genera (9). It is important to understand the molecular biology of baculovirus host range and cell type-specific restriction so that it may be possible to predict the effects of genetically modified viruses on nonhost species.The host range of baculoviruses is restricted at a stage subsequent to virus entry into cells. Free virus particles are capable of entering a wide variety of permissive and nonpermissive insect and mammalian cells, however, levels of early gene expression, DNA replication, and late-gene expression vary depending on the cell line (34, 35). In non-and semipermissive cells, including human liver cells, DNA is released into the nucleus in an expressible form (21, 35). However, in nonpermissive cells no DNA replication or late-gene expression occurs. Thus, the block to virus replication in nonpermissive insect cells appears to occur subsequent to early-gene expression.To date, s...

HCF-1 encoded by baculovirus AcMNPV is required for productive nucleopolyhedrovirus infection of non-permissive Tn368 cells

“…The hcf-1 gene of AcMNPV is one of the lef (late expression factor) genes, which is essential for viral DNA replication and expression of late genes in transient assays in Tn368, but not Sf21, cells 14 . In AcMNPV-infected Tn368 cells, the HCF-1 protein is synthesized early during infection and localizes in punctuate nuclear structures 17 , 18 . Recombinant AcMNPV defective in functional hcf-1 gene replicates normally in Sf21 cells, but exhibits various mutant phenotypes in Tn368 cells, including defective viral DNA replication and late gene transcription, and complete cessation of cellular and viral protein syntheses 15 .…”

“…In recombinant AcMNPV defective in the hcf-1 gene, however, productive infection only occurs in Sf21 cells, indicating that AcMNPV requires HCF-1 for the productive infection of Tn368 cells 14 , 15 . The HCF-1 protein contains a putative RING-finger domain, which is involved in the formation of functional HCF-1 dimers or higher-order structures in the nucleus of infected Tn368 cells 17 , 18 . It was also demonstrated that transiently expressed HCF-1 protein represses expression from the hcf-1 promoter and this repression activity of HCF-1 is required for the efficient expression of viral late genes and production of polyhedra in Tn368 cells 18 .…”

“…The HCF-1 protein contains a putative RING-finger domain, which is involved in the formation of functional HCF-1 dimers or higher-order structures in the nucleus of infected Tn368 cells 17 , 18 . It was also demonstrated that transiently expressed HCF-1 protein represses expression from the hcf-1 promoter and this repression activity of HCF-1 is required for the efficient expression of viral late genes and production of polyhedra in Tn368 cells 18 . However, the functional role of HCF-1 protein in AcMNPV-infected Tn368 cells has not been conclusively determined.…”

Baculovirus host-range

“…Mutational analysis of hcf-1 function in AcMNPV infection implicates the RING-finger domain in its ability to support virus replication and to self-associate (132).…”

“…Whether it might have a more definitive effect on larval hostrange in another host species has not been determined.It would be interesting to determine if the addition of hcf-1 to the BmNPV genome would affect its ability to infect T. ni cells. Although the specific role for hcf-1 in infection is still unclear, its activity appears to require self-association and nuclear localization (51).Hcf-1 nuclear staining is diffuse in transfected cells but shows distinct punctate foci in infected cells, suggesting that it may localize to promyelocytic leukemia (PML) nuclear bodies(132).…”