The integration of a DNA copy of an HIV-1 RNA genome into the host genome, carried out by the viral enzyme integrase, results in the formation of single-stranded gaps in cellular DNA that must be repaired. Here, we have analyzed the involvement of the PI3K kinases, ATM, ATR, and DNA-PKcs, which are important players in the DNA damage response (DDR) in HIV-1 post-integrational DNA repair (PIR). The participation of the DNA-PK complex in HIV-1 PIR has been previously shown, and the formation of a complex between the viral integrase and the DNA-PK subunit, Ku70, has been found to be crucial for efficient PIR. Now, we have shown that the inhibition of both DNA-PKcs and ATM, but not ATR, significantly reduces PIR efficiency. The activation of both kinases is a sequential process, where one kinase, being activated, activates the other, and it occurs simultaneously with the integration of viral DNA. This fact suggests that the activation of both kinases triggers PIR. Most interestingly, the activation of not only DNA-PKcs, but also ATM depends on the complex formation between integrase and Ku70. The elucidation of the interactions between viruses and DDR is important both for understanding the modulation of host cell functions by these pathogens and for developing new approaches to combat viral infections.

Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.

Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209–239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.

Cellular proteins, partners of viral enzymes, are involved in the replication of human immunodeficiency virus type I (HIV-1) at various stages. Thus, the viral enzyme integrase, participating in several stages of the viral cycle, interacts with various cellular proteins. A number of them are already known, and for some the mechanism of action has been established, but the search for cellular partners of integrase continues. In this work, the identification of cellular partners of HIV-1 integrase has been carried out by the method of cross-linking followed by mass spectrometry (XL-MS). Twelve new potential integrase partners have been identified, and some of them have been examined for their effect on the early stages of HIV-1 replication.

Human immunodeficiency virus type 1 (HIV-1) is known to activate cytosolic DNA sensor pathways, such as the cGAS-STING pathway, the activation of which leads to interferon production. The primary source of this activation is reverse transcription of the viral RNA genome into cDNA, which occurs in the cytoplasm. However, the degree of cytosolic DNA sensor activation during HIV-1 infection is significantly lower compared to that induced by DNA-containing viruses or even by the related HIV-2. This can be attributed to the successful evasion of innate immune recognition pathways by HIV-1, particularly through the disruption of cGAS-STING signaling pathway activation. In this review, we summarize the available information regarding the mechanisms employed by HIV-1 to conceal viral cDNA from cytosolic DNA sensors. Deciphering these mechanisms may reveal potential vulnerabilities that could be targeted to develop novel antiviral approaches.

Upstream open reading frames (uORFs) are widespread regulatory elements located in the 5′-untranslated regions of eukaryotic and viral mRNAs. Once considered rare exceptions to the canonical scanning model, uORFs are now recognized as actively translated features present in approximately half of human transcripts, frequently initiating at non-canonical start codons. In eukaryotes, uORFs fine-tune protein synthesis in response to cellular stress by modulating ribosome reinitiation, as exemplified by the stress-dependent regulation of ATF4 and GCN4. In viral genomes, uORFs serve as compact, evolutionarily conserved modules that optimize protein stoichiometry, coordinate expression with the host cell state, and support persistent infection across diverse viral families through cis-regulation of translation. Beyond this, uORF-encoded peptides (uPeptides) can possess independent biological activities, influencing virulence, tissue tropism, and immune modulation. In this review, we systematize the available data on the dual functionality of uORFs as cis-regulatory elements of translation and as sources of bioactive uPeptides in viruses. Deciphering the full spectrum of uORF-mediated mechanisms may reveal novel layers of viral replication control and uncover potential targets for antiviral development, although, as discussed here, the available evidence for such applications is still largely limited to proof-of-principle mutagenesis.