64 research outputs found
The Gag Cleavage Product, p12, is a Functional Constituent of the Murine Leukemia Virus Pre-Integration Complex
The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration
HIV infection of non-dividing cells: a divisive problem
Understanding how lentiviruses can infect terminally differentiated, non-dividing cells has proven a very complex and controversial problem. It is, however, a problem worth investigating, for it is central to HIV-1 transmission and AIDS pathogenesis. Here I shall attempt to summarise what is our current understanding for HIV-1 infection of non-dividing cells. In some cases I shall also attempt to make sense of controversies in the field and advance one or two modest proposals
SUMO-Interacting Motifs of Human TRIM5Ξ± are Important for Antiviral Activity
Human TRIM5Ξ± potently restricts particular strains of murine leukemia viruses
(the so-called N-tropic strains) but not others (the B- or NB-tropic strains)
during early stages of infection. We show that overexpression of SUMO-1 in human
293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This
block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor
the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1
overexpression. The block occurred prior to reverse transcription and could be
abrogated by large amounts of restricted virus. Knockdown of TRIM5Ξ± in 293T
SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral
effects, and this loss of restriction could be restored by expression of a human
TRIM5Ξ± shRNA-resistant plasmid. Amino acid sequence analysis of human
TRIM5Ξ± revealed a consensus SUMO conjugation site at the N-terminus and
three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of
the TRIM5Ξ± consensus SUMO conjugation site did not affect the antiviral
activity of TRIM5Ξ± in any of the cell types tested. Mutation of the SIM
consensus sequences, however, abolished TRIM5Ξ± antiviral activity against
N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region
of the Gag gene reduced the SUMO-1 block and the TRIM5Ξ± restriction of
N-MLV. Our data suggest a novel aspect of TRIM5Ξ±-mediated restriction, in
which the presence of intact SIMs in TRIM5Ξ±, and also the SUMO conjugation
of CA, are required for restriction. We propose that at least a portion of the
antiviral activity of TRIM5Ξ± is mediated through the binding of its SIMs to
SUMO-conjugated CA
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