Researchers reveal and image new phase of HIV infection for the first time.


How HIV-1 overcomes the person’s defenses and infects their cells has been studied intensively for over 30 years. While much has been learned, there remain events in the viral life cycle that resist interrogation. One such crucial area is the initial intra-nuclear portion of infection, from the virus’s nuclear entry to its integration into chromatin.  Now, a study from researchers at University of Massachusetts and the Duke University has identified a new life cycle stage in HIV infection, thanks to a novel technique they developed to take images of intact infected cells. The team state that their study reveals an important stage and mechanism in HIV infection that was previously unappreciated which may lead to ways to stop the virus from becoming part of the human’s DNA and infecting them for life.  The opensource study is published in the journal Cell Reports.

Previous studies show that there are certain characteristics of a virus that can only be learnt by keeping it intact and seeing it in action in single cells.  Researchers have been studying HIV for 30 years, however, without a valid way to look inside infected cells it has been difficult to gain a complete picture of what the virus is doing and how to stop it.  Up to this point, scientists have been unable to generate good images of HIV inside the nucleus using standard techniques. Because of this limitation, most insights into HIV’s transit across the nuclear membrane have been gained through indirect molecular biology and biochemistry methods that evaluate large cell populations.  Using ViewHIV, the researchers were able to closely monitor the migration of HIV, which is made up of a protein capsule or capsid that contains the virus’s DNA, as it passes through the nuclear membrane and moves around inside the nucleus.  The current study shows that this phase of infection, dubbed intra-nuclear migration relies on the human protein CPSF6 to guide the virus through the host cell’s nucleus and position it at active genes where it prefers to make its home

The current study using images produced by ViewHIV, tracked the virus and its capsid as it moved through the cytoplasm, across the nuclear membrane and finally into the nucleus where it permanently integrates into the host cell’s DNA. Results show that by knocking down certain host proteins, the impact that these proteins had on the virus’ ability to enter the nucleus and integrate into the host genome can be observed.  Data findings show that the viral capsid played an important role in the virus’s ability to enter and navigate through the nucleus.

The group note that many studies hypothesized that HIV shed it protein capsid before it enters the nuclear pore complex, however, their images clearly show that a portion of the capsid is still present and associated with the viral DNA after nuclear entry; with the final shedding of capsid occurring when the virus reaches its final destination.  Further investigation showed that it is the capsid’s use of the host proteins CPSF6 and TNPO3 which allow it to enter and navigate through the nucleus; without this help, the virus gets stranded outside or at the edge of the nucleus.

The team state that the CPSF6 protein normally works to modify the cell’s newly made messenger RNAs and its goal is to find active genes once it gets into the nucleus. Results show that when a cell is infected with HIV, the virus takes advantage of CPSF6 by hitching a ride on the protein, which is ferried across the nuclear membrane by the nuclear importer, TNPO3. Data findings show that once inside the nucleus, HIV, because it’s bound to CPSF6, is carried to active gene areas where it prefers to integrate; in the absence of TNPO3, the virus is unable to cross the nuclear membrane. The lab stress that without CPSF6, HIV is unable to find the active gene regions that it prefers for integration; instead integrating into less active regions.

The researchers explain that ViewHIV is capable of generating images of both the viral genome and protein capsid simultaneously inside an infected host cell. They go on to add that ViewHIV pairs a very sensitive type of fluorescence in situ hybridization (FISH) with a monoclonal antibody that binds to the viral capsid and allows the capsid, tagged with a fluorescent antibody, to be seen in the images of the nucleus.  Results show that this technique allows visualisation of the movement and fate of the viral capsid, DNA and RNA inside the cell.

The team surmise that their findings point to a previously undescribed state in HIV’s life cycle taking place between the time the virus enters the nucleus and the time its DNA is integrated into our genome, which was only discovered thanks to the development of ViewHIV.  For the future the researchers believe that ViewHIV is going to be a great tool for unlocking the mechanisms that govern the early state of HIV’s life cycle.  They go on to add that with their technique the global medical community can better determine how HIV establishes itself into the human DNA and develop new ways to stop that from happening.

Source: UMass Medical School Communications

Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA’s interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1’s depth of nuclear invasion, potentially aiding the virus’s integration into gene-dense regions.  Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration.  Brass et al 2015.

Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA’s interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1’s depth of nuclear invasion, potentially aiding the virus’s integration into gene-dense regions. Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration. Brass et al 2015.

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