Viruses are obligate intracellular parasites that highjack the host cell machinery to complete their replication cycle, a process that involves many protein-protein interactions between the virus and its host. During HIV-1 infection, the capsid protein (CA) –which assembles into a conical core that houses the viral genome– recruits host cell factors to facilitate critical steps in the viral replication cycle. For instance, HIV-1 CA binds to host proteins CPSF6 and CypA to facilitate HIV-1 integration into the host genome and to protect against host defenses, respectively. Predictably, single point HIV-1 CA mutants that lack binding to CPSF6: CA mutant N74D, and those that lack binding to CypA: CA mutant P90A, infect cells less efficiently. These HIV-1 CA mutants are also more sensitive to the effects of interferon (IFN), the signaling molecule that induces the expression of thousands of genes in response to viral infections –collectively known as Interferon Stimulated Genes (ISGs), from which a subset have antiviral functions and are known as restriction factors.
Due to its crucial role in infection, it is not surprising that the HIV-1 CA is the target of several capsid-targeting restriction factors, including TRIM5α and MXB. Researchers in the Emerman Lab (Human Biology) hypothesized that the IFN hypersensitivity of HIV-1 CA mutants N74D and P90A is due to restriction by one or multiple capsid-targeting ISGs. To test their hypothesis, the researchers used their in-house HIV-CRISPR screen, which identified for the first time TRIM34 as a novel HIV-1 restriction factor. Dr. Molly Ohainle, a Senior Staff Scientist and first author in the study, remarks: “Here we show that HIV capsids with single mutations are sensitive to inhibition by capsid-targeting antiviral proteins, including the newly-described HIV restriction factor, TRIM34.” The group recently published their results in PLOS Pathogens.
In their HIV-CRISPR screen, a library of HIV-CRISPR vectors that encodes for a sgRNA-Cas9 complex with the specificity to target ~2000 ISGs is used to transduce the THP-1 cell line. The resulting collection of HIV-CRISPR KO cells is pretreated with IFN and then infected with a replication-competent HIV-1 variant of interest. As the virus replicates, it packages the HIV-CRISPR vector. After a few days, the viral RNA and the genomic DNA are extracted from the supernatant and cells, respectively, and sequenced. Analysis of the data is used to calculate a score based on the relative abundance of sgRNAs present in the HIV-CRISPR vector. This score is then used to identify restriction factors because cells with a knockout of a restriction factor gene with specificity for the HIV-1 variant assayed will produce more viruses relative to a non-restrictor gene knockout.
The researchers performed the HIV-CRISPR screen using wild type HIV-1, and CA mutants N74D and P90A. When the library was infected with the CypA binding deficient P90A CA mutant, the TRIM5α gene had one of the highest scores relative to HIV-1 wild type CA. In contrast, in the screen using the CPSF6 binding deficient N74D mutant, the TRIM34 gene was a top hit. TRIM34 is a member of the large TRIM family of proteins that includes the well-characterized restriction factor TRIM5α. The TRIM5α sensitivity to the P90A mutants is in agreement with previous reports. However, these findings are the first description of TRIM34 as an HIV-1 capsid inhibitor.
To validate the inhibitory activity of TRIM34, the researchers generated cells modified to lack TRIM34 expression. They used THP-1 cells, the cell line used for the initial screen, and primary CD4+ T cells, the primary target of HIV-1 infection. Both modified and control cell types were then infected with either wild type HIV-1 CA or the HIV-1 N74D mutant, with or without pretreatment with IFN. The lack of TRIM34 did not affect replication of the wild type virus, but it rescued replication of the N74D mutant, showing that TRIM34 can specifically block replication of the CA mutant. Interestingly, they found that the rescue was independent of IFN treatment as they obtained similar results in both IFN-treated and untreated cells, suggesting that, unlike the IFN-induced TRIM5α, TRIM34 is likely always active and able to inhibit HIV-1 CA.
The researchers then asked if TRIM34 could inhibit other lentiviruses, an activity that might suggest a potential role as a species barrier for cross-species transmission. To answer this question, they overexpressed human TRIM34 in THP-1 cells and infected the cells with SIV from macaques (SIVmac) and SIV from African green monkeys (SIVagm). They observed that human TRIM34 overexpression restricted the replication of both SIVmac and SIVagm, demonstrating that TRIM34 broadly inhibits lentiviruses. “Restriction by TRIM34 may present a barrier to effective replication of HIV in human cells as well as a to cross-species transmission of primate lentiviruses into humans, a key step in the genesis of the current HIV pandemic.”, Dr. Ohainle added.
Guided by previous studies that described protein-protein interactions between TRIM34 and TRIM5α, the investigators decided to test if TRIM34 required TRIM5α to restrict HIV-1 infection. To this end, the investigators generated THP-1 cells that overexpress TRIM34 but lack TRIM5α expression. They found that –compared to control cells that continue to express TRIM5α– the THP-1 cells that lack TRIM5α lost the ability to restrict the N74D mutant, showing that TRIM34 activity is dependent on TRIM5α. To further validate this finding, they also performed similar experiments in primary CD4+ T cells and monocyte-derived dendritic cells, both physiological targets of HIV-1 infection. They knocked down either TRIM5α or TRIM34 in these cells and observed that reduction of TRIM5α or TRIM34 expression was sufficient to rescue the N74D mutant, demonstrating again that TRIM34 and TRIM5α work in the same pathway to inhibit the N74D mutant. However, knockdown of TRIM34 alone was not sufficient to rescue the P90A mutant, showing that TRIM5α restriction is independent of TRIM34.
Taken together, using their multifunctional HIV-CRISPR screen, the group identified TRIM34 as an inhibitor of HIV-1. Their validation experiments revealed that TRIM34 is a broad inhibitor of HIV-1 and SIV capsids and that its activity is dependent on TRIM5α. Because the TRIM34-mediated inhibition is independent of IFN, their screen did not identify the ISGs responsible for the hypersensitivity of CA mutants. Dr. Ohainle, on future directions from the study: “We believe that other antiviral proteins with activity against HIV and related viruses still remain to be discovered. We show here that these can be discovered using our unbiased CRISPR screening approach. Future work will be aimed at understanding the full arsenal of cellular factors that target HIV.”
This research was supported by grants from the National Institutes of Health.
UW/Fred Hutch Cancer Consortium member Dr. Michael Emerman contributed to this work.
Ohainle M, Kim K, Komurlu Keceli S, Felton A, Campbell E, Luban J, et al. 2020. TRIM34 restricts HIV-1 and SIV capsids in a TRIM5α-dependent manner. PLoS Pathog 16(4): e1008507. https://doi.org/10.1371/journal.ppat.1008507