The early steps of retrovirus replication leading up to provirus establishment are highly dependent on cellular processes and represent a time when the virus is particularly vulnerable to antivirals and host defense mechanisms. However, the roles played by cellular factors are only partially understood. To identify cellular processes that participate in these critical steps, we employed a high volume screening of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a role for 3′-phosphoadenosine 5′-phosphosulfate synthase 1 (PAPSS1), one of two enzymes that synthesize PAPS, the high energy sulfate donor used in all sulfonation reactions catalyzed by cellular sulfotransferases. The role of the cellular sulfonation pathway was confirmed using chemical inhibitors of PAPS synthases and cellular sulfotransferases. The requirement for sulfonation was mapped to a stage during or shortly after MLV provirus establishment and influenced subsequent gene expression from the viral long terminal repeat (LTR) promoter. Infection of cells by an HIV vector was also shown to be highly dependent on the cellular sulfonation pathway. These studies have uncovered a heretofore unknown regulatory step of retroviral replication, have defined a new biological function for sulfonation in nuclear gene expression, and provide a potentially valuable new target for HIV/AIDS therapy.

In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.The retroviral life cycle is a multistep process, divided into early and late stages (reviewed in reference 8). The early stages consist of virus binding to a cellular receptor, fusion of viral and cellular membranes, delivery of the viral core into the cytoplasm, reverse transcription of the positive-strand RNA genome to generate a doubled-stranded DNA product, translocation of viral nucleoprotein complexes to the nucleus, and integration of the viral DNA into the host cell genome to generate a provirus. Interaction of the viral envelope protein (Env) with cellular receptors is the primary determinant for retroviral entry (reviewed in reference 8). However, the events that occur following virus-cell membrane fusion and that lead to proviral DNA establishment, especially those involving cellular factors, are only partially understood (18). Cellular factors other than receptors that have been implicated as playing important roles at distinct early steps of retroviral replication include actin, microtubules, importin-7, HMGa1, LAP-2␣ and the barrier-to-autointegration factor (9,11,14,28,40). A putative serine kinase that phosphorylates murine leukemia virus (MLV) p12 has also been implicated in cytoplasm-to-nuc...

To identify cellular processes involved in retroviral infection, we employed a high-volume forward genetic screen of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a clonal cell line that exhibited approximately 10-fold reduced gene expression from MLV vectors following infection despite supporting normal levels of MLV reverse transcription and integration. The defect in this cell line was specific for the MLV long terminal repeat (LTR) promoter, as normal levels of reporter gene expression were obtained from both an internal cytomegalovirus (CMV) promoter contained within an LTR-defective MLV vector and LTR expression from an avian sarcoma and leukosis virus (ASLV) vector. Complementation and shRNA knockdown experiments demonstrated that the defective gene in these cells is ZASC1 (ZNF639), a transcription factor with strong links to cancer and inherited ataxias. We demonstrated that ZASC1 is a sequence-specific DNA binding protein with three closely related binding sites located within the MLV LTR promoter, but it does not bind to the ASLV promoter. Mutating these putative ZASC1 binding sites significantly reduced levels of MLV gene expression. While wild-type ZASC1 activated expression from the MLV promoter, a green fluorescent protein-ZASC1 fusion protein showed dominant-negative inhibition of MLV gene expression. These studies identify the cellular transcription factor ZASC1 as an activator of MLV gene expression and provide tools that should be useful in studying the links between ZASC1 and human diseases.The Retroviridae family includes the human pathogens human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2), the causative agents of AIDS. The study of prototypical simple retroviruses such as murine leukemia virus (MLV) and avian sarcoma and leukosis virus (ASLV) has lead to significant advances in the understanding of retroviral infections and host cell processes (10,22,33). The early stages of the retroviral replication cycle consist of virus-receptor binding, virus-cell membrane fusion, reverse transcription, nuclear translocation, and viral DNA integration into a cellular chromosome, which generates the provirus. The late stages consist of proviral transcription by host RNA polymerase II, RNA processing and cytoplasmic export, the translation of viral proteins, viral assembly, egress, and maturation. These events are heavily dependent upon host cellular machinery.Many cellular factors that regulate different steps of retroviral replication were identified previously through a combination of genetic and biochemical approaches (4,7,10,18,23,34,37). However, it is likely that other important cellular factors remain to be identified. To illustrate this point, we recently used a forward genetic approach, based upon retroviral insertional mutagenesis in CHO-K1 cells, to uncover an unprecedented role for the host cell sulfonation pathway in regulating HIV-1 and MLV gene expression (5). The role of this cellular pathway in provirus transcripti...

Transcription from the HIV-1 LTR promoter efficiently initiates but rapidly terminates because of a non-processive form of RNA polymerase II. This premature termination is overcome by assembly of an HIV-1 TAT/P-TEFb complex at the transactivation response region (TAR), a structured RNA element encoded by the first 59 nt of HIV-1 mRNA. Here we have identified a conserved DNA-binding element for the cellular transcription factor, ZASC1, in the HIV-1 core promoter immediately upstream of TAR. We show that ZASC1 interacts with TAT and P-TEFb, co-operating with TAT to regulate HIV-1 gene expression, and promoting HIV-1 transcriptional elongation. The importance of ZASC1 to HIV-1 transcription elongation was confirmed through mutagenesis of the ZASC1 binding sites in the LTR promoter, shRNAs targeting ZASC1 and expression of dominant negative ZASC1. Chromatin immunoprecipitation analysis revealed that ZASC1 recruits Tat and P-TEFb to the HIV-1 core promoter in a TAR-independent manner. Thus, we have identified ZASC1 as novel regulator of HIV-1 gene expression that functions through the DNA-dependent, RNA-independent recruitment of TAT/P-TEFb to the HIV-1 promoter.