restriction factors = paper 2

Question 1

discovery of TRIM5a

Answer 1

TRIM5a interferes with viral uncoating to block RT and productive infection
Stremlau et al (2004)
- prepared cDNA library from primary rhesus macaque lung fibroblasts, resistant to HIV infection = represented all the mRNA expressed in these cells
produced murine leukaemia virus vectors, each containing 1 cDNA from fibroblasts
transduced into HeLa cells, normally permissive to HIV-1 infection = each HeLa cell expressed 1 protein from fibroblasts
challenged HeLa cells with GFP-tagged HIV-1 = infected cells turn green, resistant cells do not
identified 2 HeLa clones resistant to infection by HIV = sequences of only cDNA insert common to both encodes TRIM5a

Question 2

discovery of APOBEC3G

Answer 2

packaged into budding viruses, facilitates C –> U deamination + G –> A hypermutation to compromise coding/replicative capacity of HIV

Sheehy et al (2002)
- previously observed that HIV1 lacking Vif shows differential replication in certain cells (permissive in CEM-SS T cell line, non-permissive in primary T cells and CEM lines)
used cDNA substraction strategy to identify RF responsible
- extracted RNA from non-permissive CEM cells + converted to cDNA
- mixed with excess of cDNA from CEM-SS permissive and allowed cDNA to hybridise
- subtracted unhybridised fraction, enriched in sequences only found in non-permissive cells
- amplified by PCR
- Used subtracted cDNAs as probes in Northern blotting of RNAs extracted from a panel of permissive and non-permissive cells
• Found CEM15 = present in all non-permissive cell lines, absent from all permissive cell lines
• Ectopic expression of CEM15 in permissive cells inhibited infectivity of Vif-deficient HIV-1, but not WT HIV = Vif inhibits CEM15

• Sequence homology to APOBEC1 = identified enzyme as cytidine deaminase

Question 3

discovery of tetherin

Answer 3

• Neil et al (2008) = identified tetherin through comparative transcriptomics
- looked at levels of mRNA from different cell types by microarray
- looked for genes with constitutive expression in cells in which Vpu is required for infectivity (e.g. HeLa), low expression in cell lines where Vpu is dispensable (e.g. HOS) and induced by IFN treatment in 3 other cell lines
- identified tetherin = expression > 20-fold greater in HeLa than HOS< induced >20 fold by IFN-a in 293T cells
• also used EM to look at the appearance of surface of infected cells expressing tetherin or not
- cells not expressing tetherin = few virus particles visible as viruses have all budded off
- cells expressing tetherin = lots of virus particles tethered to cell surface, as they cannot bud off properly