and in vivo. Adenovirus enters cells by receptor-mediated endocytosis, reaches the cytosol by acid-assisted disruption of the endosomal membrane, translocates as a naked particle along stable microtubules to the nucleus, disassembles and imports its DNA genome into the nucleoplasm: Have a look to a these detailed models created by Urs Greber at the University of Zurich (Switzerland):

A detailed infection-model of Ad 2; A more detailed model of the Virus-nuclear pore complex (NPC) association and capsid disassembly and a more presize model for the nuclear pore complex-dependent Ad2 capsid disassembly and DNA import here:

Replicative Cycle
The adenovirus replicative cycle is divided into early and late phases with the late phase occurring when viral DNA replication begins. Normally structural proteins are the major polypeptides synthesized during the late phase. This division into early and late is over-simplified and somewhat misleading as we will see in upcoming revelations. After infection adenoviruses rapidly shutdown host cell macromolecular synthesis i.e. cell DNA and protein synthesis. The mechanism of shutoff is not clear but protein synthesis is rapidly inhibited whereas cell DNA synthesis is shut down at a more leisurely pace. The adenovirus lytic cycle is very efficient producing 10,000 virions per cell with one virus cycle occurring in 32-36 hours.

Early Transcription
Viral gene expression is not a haphazard process. On the contrary it is highly ordered and carefully orchestrated in much the same manner as genes are regulated for cell division or embryonic deve-lopment. The ability to function efficiently is critical to the virus's success in a hostile environment where it is dependent on the host cell for basic macromolecular machinery and supplies. RNA polymerase II is responsible for viral gene transcription at both early and late times. Early RNA synthesis occurs from at least 7 distinct regions of the viral genome, from separate promoters and from both DNA strands. Extensive splicing occurs for both early and late transcripts, an obser-vation that was first documented in adenoviruses. Recent evidence has led to further qualification and subdivision of early transcription. Studies with the protein synthesis inhibitors cycloheximide and anisomycin have shown a defined order of mRNA expression.

• immediate early -- L1 · pre-early -- E1A · delayed early -- E1B, E2A, E2B, E3, E4 · intermediate -- IVa2, IX · Rightward transcription. · E1A - transformation, viral gene transcription · E1B - transformation · E3 - nonessential in tissue culture, immune modulation in vivo? · L1 - function unknown Leftward transcription. · E4- immune modulation · E2A - DNA binding protein (DBP) · E2B - DNA polymerase (140kd) and 80 kd precursor to terminal · protein (pTP)
  

Regulation of early gene expression. The E1A region is pivotal in early gene synthesis. The major product of the E1A is a 13S mRNA which encodes a 289 amino acid protein with diverse func-tions including the induction of DNA synthesis, induction of mitosis (transformation) and transacti-vation of viral genes. E1A controls E1B, E2, E3, and E4 mRNA accumulation but cannot itself bind DNA. E1A products appear to activate a series of host proteins which bind to target sequences within early gene promoters.

Early proteins
Tumor antigens produced in hamsters bearing adenovirus induced tumors were the first source of information on early proteins. Antisera from these animals reacted with adenovirus infected cells. Since early regions E1A and E1B are the only areas commonly present in tumor cells this led to the early characterization of proteins from these two regions. Subsequent studies employing mRNA hybrid selection and in vitro selection provided more definitive locations for many of the early proteins.

Late Transcrption
Control of late transcription is very complex and little is known about the molecular mechanisms involved in regulating the switch from early to late gene expression. One of the major features of late transcription is the presence of a tripartite leader for late mRNA. Synthesis of late transcripts begin at 16.45 map units and extend to 99 map units. Extensive splicing of this large transcript then occurs. In general their are 5 classes of late transcripts which have variable 5'ends and coterminal 3' ends.
VA RNAs. Virus associated RNAs known as VA RNA I and VA RNA II are small (155 bases) RNAs generated by pol III. They do not encode polypeptides and have the potential to form extensive secondary structure. Suggested functions for these RNAs particularly VA I include: 1. regulation of late mRNA splicing; 2. control of the rate of translation of late polypeptides and 3. inhibition of the effects of interferon by blocking binding of dsRNA to cellular protein kinase and by binding the kinase and blocking eIF2 inactivation.

Late proteins
The synthesis of late proteins is maximal 20 hr post-infection. Most late proteins are structural components of the virion but some early genes are also made late.

· Techniques of note:
· Hybrid selection\ in vitro translation
· Hybrid arrested translation (HART)
· heteroduplex analysis
· S1 nuclease mapping
· in vitro replication
· UV mapping of transcripts

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