Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, onis and orML. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on onis, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described (14), and the recombinant plasmid was used to generate the recombinant baculovirus AcNPV/UL9 as described (15,16).Antisera. Decapeptides corresponding to the predicted carboxyl-terminal amino acid sequence ofHSV-1 genes UL5, UL8, UL9, and UL52 were purchased from Biosearch (San Rafael, CA) and were covalently coupled to keyhole limpet hemocyanin. Approximately 0.5 mg of coupled peptide was used to immunize rabbits biweekly for a total of three injections. Sera were collected at biweekly intervals beginning one month after the first injection. A complete characterization of these sera will be published elsewhere.Preparation of Baculovirus-Infected Cell Extract. Fifteen 150-cm2 flasks of nearly confluent SF9 cells were infected with the recombinant virus AcNPV/UL9 at a multiplicity of infection of 10-20 plaque-forming units per cell. After 54 hr at 28°C, the cells were dislodged from the flasks by shaking and were washed with phosphate-buffered saline. NucleiAbbreviations: HSV, herpes simplex virus; AcNPV, Autographa californica nuclear polyhedrosis virus.

In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oris). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.The 153-kilobase genome of herpes simplex virus 1 (HSV-1) contains both cis-and trans-acting elements that function in viral DNA replication (1). The cis-acting elements correspond to the origins of DNA replication (oris and oriL) (2-4), and the trans-acting elements very likely code for most and possibly all of the enzymes required for HSV-1 DNA replication. The nucleotide sequence of the entire HSV-1 genome has been determined (5), allowing assignment ofHSV-1 genes and their products to specific open reading frames. Seven of these open reading frames have been shown to be necessary and sufficient for the replication in trans of plasmids containing either origin of DNA replication, oriL or oris (6).These open reading frames also correspond to seven complementation groups known to be essential for HSV-1 DNA replication (7)(8)(9). Of the seven open reading frames, three have thus far been identified and shown to encode the herpes DNA polymerase (Pol) (10), a single-stranded DNA-binding protein (ICP8) (11), and the oris-binding protein (UL9) (12).A double-stranded DNA-binding protein whose role in DNA replication is unknown is encoded by the fourth open reading frame (UL42) (13).In this report we show that the HSV-1-induced DNA helicase that we have identified (14) consists of three polypeptides encoded by the three remaining open reading frames UL5, UL8, and UL52. We have also found that a DNA primase activity is tightly associated with the three-subunit enzyme, establishing the presence of an HSV-1-encoded helicase-primase complex in HSV-1-infected cells.

Mitochondrial DNA (mtDNA) is unusual in its rapid rate of evolution and high level of intraspecies sequence variation. The latter is thought to be related to the strict maternal inheritance of mtDNA, which effectively isolates within a species mitochondrial gene pools that accumulate mutations and vary independently. A fundamental and as yet unexplained aspect of this process is how, in the face of somatic and germ-line mtDNA ploidy of 10(3) to 10(5) (refs 4, 5), individual variant mtDNA molecules resulting from mutational events can come to dominate the large intracellular mtDNA population so rapidly. To help answer this question, we have determined here the nucleotide sequence of all or part of the D-loop region in 14 maternally related Holstein cows. Four different D-loop sequences can be distinguished in the mtDNA of these animals. One explanation is that multiple mitochondrial genotypes existed in the maternal germ line and that expansion or segregation of one of these genotypes during oogenesis or early development led to the rapid genotypic shifts observed.

SummaryThe use of influenza A virus-inducible reporter gene segments in detecting influenza A virus replication was investigated. The RNA polymerase I promoter/terminator cassette was used to express RNA transcripts encoding green fluorescence protein or firefly luciferase flanked by the untranslated regions of the influenza A/WSN/33 NP segment. Reporter gene activity was detected after reconstitution of the influenza A virus polymerase complex from cDNA or after virus infection, and was influenza A virus-specific. Reporter gene activity could be detected as early as 6 hours post infection and was virus dose-dependent. Inhibitory effects of antibodies or amantadine could be detected a nd quantified rapidly, providing a means of not only identifying influenza A virus -specific replication, but of determining the antigenic subtype as well as antiviral drug susceptibility. Induction of virus-specific reporter genes provides a rapid, sensitive method for detecting virus replication, quantifying virus titers and assessing antiviral sensitivity as well as antigenic subtype.

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Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.

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Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.