Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection.

In mammalian cells, a conserved multiprotein complex of Mre11, Rad50 and NBS1 (also known as nibrin and p95) is important for double-strand break repair, meiotic recombination and telomere maintenance. This complex forms nuclear foci and may be a sensor of double-strand breaks. In the absence of the early region E4, the double-stranded DNA genome of adenovirus is joined into concatemers too large to be packaged. We have investigated the cellular proteins involved in this concatemer formation and how they are inactivated by E4 products during a wild-type infection. Here we show that concatemerization requires functional Mre11 and NBS1, and that these proteins are found at foci adjacent to viral replication centres. Infection with wild-type virus results in both reorganization and degradation of members of the Mre11-Rad50-NBS1 complex. These activities are mediated by three viral oncoproteins that prevent concatemerization. This targeting of cellular proteins involved in genomic stability suggests a mechanism for 'hit-and-run' transformation observed for these viral oncoproteins.

The maintenance of genome integrity requires a rapid and speci®c response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G 2 / M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells. Keywords: adenovirus/ATM/ATR/DNA double-strand breaks/Mre11 complex Introduction A central player in the cellular response to DSBs is the Mre11 complex, consisting of Mre11, Rad50 and NBS1 (Xrs2 in yeast) (Petrini, 1999;D'Amours and Jackson, 2002). Hypomorphic mutations in the genes for Mre11 and NBS1 result in the human genetic instability diseases ataxia-telangiectasia like disorder (A-TLD) and Nijmegen breakage syndrome (NBS), respectively (Carney et al., 1998;Stewart et al., 1999). Cell lines derived from these patients are sensitive to ionizing radiation (IR) and exhibit radioresistant DNA synthesis (RDS). Cytologic and genetic evidence suggests that the Mre11 complex acts as a DNA damage sensor (Mirzoeva and Petrini, 2001;Usui et al., 2001), but de®nitive evidence in mammalian cells has thus far remained elusive.The related PI3-like kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), are signal transducers of the DNA damage response. Mutations in ATM lead to ataxia-telangiectasia (A-T), and cells from these patients are hypersensitive to DSBs and defective for induction of the G 1 /S, intra-S and G 2 /M cell-cycle checkpoints (Shiloh, 2003). Null mutations of ATR are lethal, but the protein has been studied using a conditional knockout allele or dominant-negative kinase dead mutants, which sensitize cells to all forms of DNA damage and affect the intra-S and G 2 /M checkpoint responses ( We have demonstrated that adenovirus (Ad) avoids detrimental genome concatemerization through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins (Stracker et al., 2002). Here we show for the ®rst time that infection with an adenovirus lacking the E4 region induces a cellular DNA damage response that requires ATM and ATR. We identi®ed mutants of E1b55K that can selectively degrade different substrates. Using these E1b55K mutants we show that the Mre11 complex is required for the activation of ATM and ATR in response to Ad infection. In addition, we show that Mre11 degrad...

APOBEC3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and transposition of endogenous retroelements. One family member, APOBEC3A (hA3A), is an orphan, without any known antiviral activity. We show that hA3A is catalytically active and that it, but none of the other family members, potently inhibits replication of the parvovirus adeno-associated virus (AAV). hA3A was also a potent inhibitor of the endogenous LTR retroelements, MusD, IAP, and the non-LTR retroelement, LINE-1. Its function was dependent on the conserved amino acids of the hA3A active site, consistent with a role for cytidine deamination, although mutations in retroelement sequences were not found. These findings demonstrate the potent activity of hA3A, an APOBEC3 family member with no previously identified function. They also highlight the functional differences between APOBEC3 proteins. The APOBEC3 family members have distinct functions and may have evolved to resist various classes of genetic elements.

Broadly defined, the concept of gene therapy involves the transfer of genetic material into a cell, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient. A key factor in the success of gene therapy is the development of delivery systems that are capable of efficient gene transfer in a variety of tissues, without causing any associated pathogenic effects. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, currently offer the best choice for efficient gene delivery. Their performance and pathogenicity has been evaluated in animal models, and encouraging results form the basis for clinical trials to treat genetic disorders and acquired diseases. Despite some initial success in these trials, vector development remains a seminal concern for improved gene therapy technologies.