A highly sensitive single-round infection assay using a bacterial chloramphenicol acetyltransferase was developed to analyze an early stage of human immunodeficiency virus type 1 replication. By a combination of transfection and single-round infection assay, a virus with a vifmutation, depending on host cells from which the virus was derived, was demonstrated to be defective at the early phase of infection cycle. Analysis of viral proteins synthesized in cells indicated that incorporation of the Env surface protein into virions of the vif mutant, again in a cell-dependent way, was greatly restricted. Taken together, it is concluded that the Vif protein acts through modulation of the Env protein in the virions, directly or indirectly, to enhance viral infectivity in a certain cell type. Of accessory genes (4) of human immunodeficiency virus type 1 (HIV-1), the vifgene is essential for virus growth in a

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.

Human papillomaviruses (HPVs) target the stratified epidermis, and can causes diseases ranging from benign condylomas to malignant tumors. Infections of HPVs in the genital tract are among the most common sexually transmitted diseases, and a major risk factor for cervical cancer. The virus targets epithelial cells in the basal layer of the epithelium, while progeny virions egress from terminally differentiated cells in the cornified layer, the surface layer of the epithelium. In infected basal cells, the virus maintains its genomic DNA at low-copy numbers, at which the viral productive lifecycle cannot proceed. Progression of the productive lifecycle requires differentiation of the host cell, indicating that there is tight crosstalk between viral replication and host differentiation programs. In this review, we discuss the regulation of the HPV lifecycle controlled by the differentiation program of the host cells.

The genetic and functional basis of the replicationdefective nature of human immunodeficiency virus type 1 (HIV-1) in monkey cells was studied. By the generation and characterization of chimeras between HIV-1 and simian immunodeficiency virus, the sequence encompassing the 3' half of the long terminal repeat, gag and pol genes of HIV-1 was found to be responsible for the growth restriction. Early and late phases of HIV-1 replication in monkey cells were analysed in detail using several assay systems: transfection/coculture, transcomplementation between various proviral clones carrying the CAT gene and effector clones and evaluation of transcription and reverse transcription. All the data were consistent with the notion that replication is blocked at a very early stage(s) such as uncoating and/or reverse transcription in monkey cells.

The papillomavirus E2 gene product plays a pivotal role in viral replication. E2 has multiple functions, including (i) transcriptional activation and repression of viral promoters and (ii) the enhancement of viral DNA replication. It was previously reported that E2 suppressed the growth of papillomavirus-positive cervical carcinoma cell lines. In the present study, we investigated the mechanisms of E2 growth inhibition. We found that the transcriptional activation function of E2 is required for inhibition of the growth of HeLa cells as well as for transcriptional repression of the viral E6/E7 promoter. It had been previously postulated that transcriptional repression of the E6/E7 promoter results from E2 binding its cognate sites proximal to the E6/E7 promoter and displacing other cellular transcriptional factors. In this study, we report a requirement for the transcription activation function for the binding of E2 to transcriptionally active templates.The papillomavirus replication cycle is regulated by the viral E2 protein, a sequence-specific DNA binding protein (1,35,53). Depending on the promoter context, E2 can act either as a transcriptional activator or as a repressor of viral gene expression. The promoters for E6/E7 gene expression of human papillomavirus type 16 (HPV16) and HPV18 are negatively regulated by E2. This repression is thought to be mediated by the binding of E2 to its recognition sites within the promoter and the displacement of cellular transcriptional factors from the promoter (3,12,14,15,20,23,28,41,42,54,55,58,59,61,62). E2 is also involved in the regulation of viral DNA replication through its association with E1, the viral replication factor (36,50,63,65,66,67,68). The conserved N-terminal domain of E2 is required for transactivation (TA), E1 binding, and DNA replication functions. The conserved C-terminal domain forms a dimer and functions as a DNA binding domain. Both conserved domains are linked by a hinged region (reviewed in reference 24).The loss of E2 expression has been also implicated in the development of HPV-induced carcinoma. Most human cervical carcinoma cells contain integrated HPV DNA and actively express E6/E7 genes (2, 52, 69). The E2 gene is frequently disrupted as a consequence of the integration of the viral genome, and it has been postulated that the loss of E2 somehow contributes to carcinogenic progression (9,40,47,64). E6/E7 genes are invariably expressed in HPV-positive cancers and are considered to be involved in the development of HPVassociated cancers. E6 targets the ubiquitination and proteolysis of p53 through its association with the ubiquitin protein ligase, E6AP (25,45,46). E7 binds pRB and inactivates its tumor suppressor function (17,38). Although E6 and E7 may have additional functions and cellular targets, it is believed that their inactivation of these important tumor suppressor proteins is critical for HPV-associated carcinogenesis. As mentioned above, E2 has the ability to suppress E6/E7 expression; thus, disruption of the E2 gene results in the...