Over 2.5 billion people live in areas at high risk for Dengue and related flavivirus infections (1). Dengue hemorrhagic fever, a severe complication present in ϳ5% of cases, claims more lives annually than all other hemorrhagic fevers combined (2); much of the damage is caused by death of infected cells. The fate of infected cells depends on cell type. Although flavivirus induces apoptosis of neurons and macrophages, infected hepatocytes and epithelial cells do not die. We find that flavivirus up-regulates autophagy in MDCK 3 renal epithelial cells and other cell types and subsequently protects them. We further identify nonstructural protein NS4A of both Dengue-2 and Modoc viruses as the sole viral mediator of autophagy up-regulation and protection against death. Up-regulation of autophagosomes by either live virus infection or NS4A expression depends on PI3K and is important for replication of flavivirus in renal epithelial cells. Flaviviruses often persist in liver and kidney following the acute phase of infection, suggesting that infected cells evade destruction by the host immune response, likely by flavivirusinduced up-regulation of autophagy in these cells. In vitro infection of hepatocytes and fibroblasts leads to up-regulation in autophagy and protection against death (3, 4). More relevant, Dengue-2 viruses replicate within the hepatocyte autophagosomes (5), and inhibition of autophagy attenuates virus replication (3). We extend these findings by identifying the nonstructural viral protein NS4A as the virus-encoded protein that up-regulates autophagy and thus protects the host cell against death, providing a well protected host cell for long term replication of virus.The flavivirus genome contains 10 genes encoding an ϳ11-kDa polyprotein precursor that binds to the ER membrane after translation at the rough ER as follows: three structural proteins (capsid (core) protein, pre-membrane protein ,and envelope glycoprotein) and seven nonstructural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that mediate viral replication, assembly, and evasion of the host immune system. The active proteins are cleaved from the ER-bound viral polyprotein by cellular proteases signalase and furin and viral protease NS3/ 2B. NS3 is both a viral protease (with required cofactor NS2B (6)) and a viral ATP-dependent helicase (7, 8); NS5 is an RNAdependent RNA polymerase and methyltransferase (9) responsible for virus genome replication, whereas NS1 is possibly a part of the viral replication complex (10). The small hydrophobic flavivirus proteins (NS2A, NS4A, and NS4B) remain the most poorly characterized. NS2A is required for the assembly of new flavivirus virions; NS4A associates with the virus replication complex and induces ER membrane rearrangements (discussed below), and NS4B is an antagonist of interferon. Flavivirus NS4A is an ϳ16-kDa membrane-associated protein consisting of four transmembrane helices and an N-terminal cytosolic region. Once the viral genome is translated in the ER,

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1’s lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins’ entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription.