week 3 Flashcards
2 methods for dna strand synthesis and which viruses
replication fork copies both strands at the same time with one lagging and one leading strand. always require rna primer. used by papillomaviruses, polyomaviruses, herpes and retrovirus
- strand displacement copies one strand at a time and never requires an rna primer. used by adeno and parvoviruses
papovirus - SV40 life cycle
- Attachment and entry
- Genome release
- Early RNA transcripts
- cell RNA pol II - Alternative RNA splicing
- Early protein translation
- Large T protein moves
to nucleus and binds Ori - Viral DNA replication
- Late RNA transcripts
- Large T + RNA pol II - Late RNA moves to
cytoplasm - Structural protein
translation (VP1 - VP3) - VP1 - 3 move to nucleus
- Virion assembly
- Particle release.
SV40 large T
large T is a protein transcribed from the early promoter. it enters the nucleus and binds ori to unwind the dna, allowing the binding if cellular single stranded DNA binding protein and replication protein A leading to further unwinding and access of DNA polymerase. there are repressor domains in the late promoters which are bound to cellular inhibitory binding proteins (ibp). when DNA replication begins, the concentration of ibp compared to DNA becomes diluted and the late genes will start to become expressed. this is known as antirepression.
SV40 replication
uses cellular DNA polymerase. rna primers are created by cellular dna polymerase sigma. a loop of template is thought to keep the lagging strand in place.
solution to end replication problem
the lagging strand needs multiple primers to create okazaki fragments. when the primer at the furthest end of a linear DNA molecules lagging strand is removed there is no way to fill the gap.
- SV40 has a circular genome which avoids this problem
- herpesvirus: becomes circular prior to replication. creates a long continouus template with multiple DNA joined together which are used as template for other strand and cleaved after encapsidation.
- adenoviruses use a protein primer: pre-TP (terminal protein) binds to DNA polymerase
- use fold back initiation: complimentarity in end sequences known as inverted terminal repeats (ITR) to form haairpins that can be used as primers. used by parvoviruses
- fold back initiation but in cytoplasm: poxviruses that forms a mininuclei in cytoplasm with viral enzymes
hepatitis B structure
circular dsDNA with onboard DNA polymerase. has 4 promoters which all use the same terminator. there are overlapping reading frames. hepatitis produces both subgenomic rnas that encodes proteins and pregenomic rna that serves as template for RT. in the nucleus, the incomplete circular DNA becomes filled and becomes “covalently closed circular DNA episome” in the nucleus.
reverse transcription hep B
the pregenomic RNA forms RNA structures that is recognized by RT. has identical structures on 3´ and 5´ because the pregenomic RNA is transcribed from more than 1 lap around circular dna. it jumps form one to the other and transcribes. newly formed dna is cleaved off, leaving a little bit left that serves as a primer for the synthesis of +DNA strand.
hbv life cycle
- Attachment (NTCP-R)
- Uptake
- DNA gap repair by P
- Covalently closed
circular (CCC) DNA - RNA transcription
from - DNA - RNA transport
- HBsAg translation
- Pregenome translation
- polymerase (P) - capsid
- Pregenome packaging
- Reverse transcription by P
- Nucleocapsid matures
- CCC DNA amplification
- Envelope addition
- Particle assembly in multi-vesicular bodies
- Particle release together with small VLP
gene products of simple retrovirus
env: surface and transmembrane proteins for envelope
gag: matrix protein, nucleocapsid and capsid
pol: RT and integrase
retrovirus genome
two copies of +ssRNA loosely bound together by RNA-RNA interactions. each is bound to a tRNA. has a 5´cap and a poly A tail. gag-pol forms polyprotein sometimes because slippery sequence that cause frame shift. a high degree of sequence variability exists for gag and env.
retrovirus replication
all retroviruses reverse transcribe their rna into dsDNA that they integrate into the host genome. new viral +ssRNA can only be replicated from the integrated dsDNA provirus using the host cell RNA transcription enzyme rnapol2.
1. reverse transcription occurs in the cytoplasm in a core. the dsDNA is then delivered to the nucleus where the integrase enzyme catalizes its random integration into host DNA.
2. +ssRNA is transcribed from provirus. unspliced rna is transported to ribosomes and translated into gag and gag-pol previrsor proteins. some unspliced rna moves to the plasma membrane for assembly into new virio. spliced rna is translated to ennv glycoprotein on ER and is processed in golgi.
reverse transcription of viral genomic rna ro proviral dna….
- causes a duplication of the untranslated sequences (R, U5, and U3) at the ends
of the genomic (+)ssRNA - generates a dsDNA structure called the “long terminal repeat” or LTR that is
common in mammalian DNA - Highly error prone process generating mutations
1 error in every 10,000nt - RT enzyme can switch between 2 different genomes to generate a
recombinant retrovirus genome.
human retroviriuses
- human immunodeficency virus (HIV)
- human t-cell leukemia virus (HTLV): transmits by virus infected cells. leads to proliferation of t cells from expressed viral products “zombie t cells”. direct tumor induction.
HIV
major target of infection is activated CD4+ T cells. RT is the most important drug target. when provirus is integrated, the sequences at the ends are duplicated forming LTRs which acts as the promoter for the HIV gene in human genome. responds to cellular proteins made during t cell immune activation to dramatically increase HIV expression.
HIV proteins
env: surface and transmembrane proteins for envelope
gag: matrix protein, nucleocapsid and capsid
pol: RT and integrase
the tat protein binds the TAR RNA-element and rnapol2 to promote transcriptional elongation. the rev protein stabilizes and transports unspliced rna to the cytoplasm. there are also some additional proteins such as vif, vpr, vpu and nef that are important for in vivo pathogenesis
- vif: promotes infectivity of cell free virus, blocks cell defences
- vpr: virion protein for nuclear import of cDNA and causes cell growth arrest
- vpu: regulator of particle release and env processing. prmotes HGC-1 and CD5 degradation
nef: down modulates cell MHC-1 and CD4e
