Publication Date

2019-04-10

Availability

Open access

Embargo Period

2019-04-10

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2019-03-20

First Committee Member

Enrique Mesri

Second Committee Member

Mario Stevenson

Third Committee Member

Ronald Desrosiers

Fourth Committee Member

Derek Dykxhoorn

Fifth Committee Member

Savita Pahwa

Sixth Committee Member

Susana Valente

Abstract

HIV/AIDS continues to cause significant morbidity and mortality across the globe. Even though current drug therapy can reduce the disease burden, a cure to HIV-1 infection remains elusive due to the relatively stable viral reservoir. Infected macrophages are of particular importance to the viral reservoir as they are long-lived cells that are often located in immune privileged sites and resistant to the cytopathic effects of the virus. Invasion of the cell by the primate lentiviruses HIV/SIV, is opposed by cellular restrictions including APOBEC3G, Tetherin/BST2, and SAMHD1. SAMHD1 is a dNTP triphosphohydrorylase that reduces cellular dNTP levels in non-mitotic cells, such as macrophages, where high levels of dNTPs are excessive to need. Since dNTPs are required for reverse transcription, HIV-2 and most SIVs encode a Vpx protein that promotes the proteasomal degradation of SAMHD1. This is particularly important for the infection of myeloid lineage cells in which SAMHD1 is highly expressed. It is unclear how HIV-1, which does not encode a Vpx protein, is able to evade SAMHD1 restriction. The study presented here begins by demonstrating that HIV-1 efficiently infects macrophages. Monitoring the amount of productive viral infection demonstrates how HIV-1 is not restricted in macrophages in comparison to SIVΔVpx. Next, it was demonstrated by analysis of both western blot and flow cytometry that HIV-1 infection did not affect levels of SAMHD1, in stark contrast to SIVwt infection which rapidly depleted it. Both the quantification of cellular dNTP levels and pre-infection experiments confirmed that HIV-1 does not affect the antiviral environment in macrophages. In contrast, SIVwt significantly increased dNTP levels, and SIVwt pre-infection was able to rescue SIVΔVpx and boost HIV-1 infection. Finally, SAMHD1 knock-down by siRNA validated that SAMHD1 was indeed the main restriction factor blocking SIVΔVpx infection and that SAMHD1 also affects HIV-1, though not to the same extent. In order to address how HIV-1 infects macrophages without modulating SAMHD1 or the antiviral environment, chimeric HIV/SIV variants in which the reverse transcriptases (RT) were exchanged, were created and tested for infectivity. These novel recombinant viruses were first tested for infectivity in the control cell line sMAGI, which confirmed the functional exchange of genes. Next, macrophage infection revealed that RT-SHIV remained Vpx-dependent like SIV and that RT-HSIV maintained Vpx-independence like HIV-1. This study demonstrates that the differential sensitivity of HIV-1 and SIV to SAMHD1 restriction is not fully dictated by RT but by a cis-acting determinant in the HIV-1 genome that confers intrinsic resistance to SAMHD1. Our study reveals fundamental differences between HIV-1 and SIV in the strategy used to overcome restriction by SAMHD1. The fact that HIV-1 has acquired a level of SAMHD1 resistance underscores an important role for myeloid cells in the interplay between HIV-1 and its host. Furthermore, understanding how these cellular restrictions antagonize viral replication will be important for the design of novel antiviral strategies that harness the antiviral potential of these cellular restrictions.

Keywords

HIV-1; SIV; SAMHD1; Vpx; Macrophage; SHIV

Share

COinS