Oncogenic Effects of HIV-1 Proteins, Mechanisms Behind

Funding Information: Funding: This study was supported the Russian Fund for Basic Research grants 17_54_30002 and 20‐04‐01034 to M.I., Latvian Science Council grants LZP‐2018/2‐0308 and LZP‐2020/2‐0376 to M.I., Funding Information: and NCI R01CA 217715 to J.P.The work of Francesca Chiodi is supported by a grant from the Swe‐ dish Medical Research Council (Francesca Chiodi; Vetenskapsrådet 2019‐01169). Publisher Copyright: © 2021 by the authors.People living with human immunodeficiency virus (HIV-1) are at increased risk of developing cancer, such as Kaposi sarcoma (KS), non-Hodgkin lymphoma (NHL), cervical cancer, and other cancers associated with chronic viral infections. Traditionally, this is linked to HIV-1-induced immune suppression with depletion of CD4+ T-helper cells, exhaustion of lymphopoiesis and lymphocyte dysfunction. However, the long-term successful implementation of antiretroviral therapy (ART) with an early start did not preclude the oncological complications, implying that HIV-1 and its antigens are directly involved in carcinogenesis and may exert their effects on the background of restored immune system even when present at extremely low levels. Experimental data indicate that HIV-1 virions and single viral antigens can enter a wide variety of cells, including epithelial. This review is focused on the effects of five viral proteins: envelope protein gp120, accessory protein negative factor Nef, matrix protein p17, transactivator of transcription Tat and reverse transcriptase RT. Gp120, Nef, p17, Tat, and RT cause oxidative stress, can be released from HIV-1-infected cells and are oncogenic. All five are in a position to affect “innocent” bystander cells, specifically, to cause the propagation of (pre)existing malignant and malignant transformation of normal epithelial cells, giving grounds to the direct carcinogenic effects of HIV-1.Peer reviewe

ATM and atr expression potentiates hbv replication and contributes to reactivation of hbv infection upon DNA damage

Funding Information: Funding: This work was performed within RSF grant no. 16-15-10426. Funding Information: National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, Moscow 127994, Russia; [email protected] (S.B.); [email protected] (V.C.) Institute of Immunology, Federal Medical Biological Agency, Moscow 115522, Russia NF Gamaleya Research Center of Epidemiology and Microbiology, Moscow 123098, Russia; [email protected] (E.B.); [email protected] (I.G.); [email protected] (M.I.) Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, Moscow 108819, Russia Sechenov First Moscow State Medical University, Moscow 119146, Russia; [email protected] Department of Pathology, Riga Stradins University, LV-1007 Riga, Latvia Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 76 Stockholm, Sweden Izmerov Research Institute of Occupational Health, Gene Engineering and Biotechnology, Moscow 105275, Russia; [email protected] (I.G.); [email protected] (F.U.); [email protected] (A.N.) Central Research Institute of Epidemiology, Moscow 111123, Russia Correspondence: [email protected] (A.K.); [email protected] (D.K.) Publisher Copyright: © 2019 by the authors.Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.publishersversionPeer reviewe

Cellular immune response induced by dna immunization of mice with drug resistant integrases of hiv-1 clade a offers partial protection against growth and metastatic activity of integrase-expressing adenocarcinoma cells

Funding Information: Funding: Experiments were supported by the grants of the Russian Science Fund 15-15-30039, Russian Fund for Basic Research 20-04-01034, Latvian Science Fund LZP 2018-2-03-08, and EU-ROPARTNER project “Strengthening and spreading international partnership activities of the Faculty of Biology and Environmental Protection of University of Lodz, Poland, for interdisciplinary research and innovation”. Mobility and method acquisition were supported by Swedish institute PI project 19806/2016TP, and Horizon 2020 project VACTRAIN#692293. MI and BW were supported by Horizon 2020 grant EAVI contract N68113. Funding Information: Experiments were supported by the grants of the Russian Science Fund 15-15-30039, Russian Fund for Basic Research 20-04-01034, Latvian Science Fund LZP 2018-2-03-08, and EU-ROPARTNER project ?Strengthening and spreading international partnership activities of the Faculty of Biology and Environmental Protection of University of Lodz, Poland, for interdisciplinary research and innovation?. Mobility and method acquisition were supported by Swedish institute PI project 19806/2016TP, and Horizon 2020 project VACTRAIN#692293. MI and BW were supported by Horizon 2020 grant EAVI contract N68113. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.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.publishersversionPeer reviewe

HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist

Funding Information: https://orcid.org/0000-0002-6160-2203 Bayurova Ekaterina [email protected] 1 2 Jansons Juris [email protected] 3 4 Skrastina Dace [email protected] 3 4 https://orcid.org/0000-0002-4980-9754 Smirnova Olga [email protected] 5 Mezale Dzeina [email protected] 3 Kostyusheva Anastasia [email protected] 6 Kostyushev Dmitry [email protected] 6 Petkov Stefan [email protected] 7 Podschwadt Philip [email protected] 7 https://orcid.org/0000-0003-0365-570X Valuev-Elliston Vladimir [email protected] 5 Sasinovich Sviataslau [email protected] 7 https://orcid.org/0000-0003-2278-4451 Korolev Sergey [email protected] 8 Warholm Per [email protected] 9 https://orcid.org/0000-0002-2260-6551 Latanova Anastasia [email protected] 1 5 https://orcid.org/0000-0003-2183-0858 Starodubova Elizaveta [email protected] 1 5 https://orcid.org/0000-0001-8506-2339 Tukhvatulin Amir [email protected] 1 Latyshev Oleg [email protected] 1 Selimov Renat [email protected] 10 Metalnikov Pavel [email protected] 10 Komarov Alexander [email protected] 10 https://orcid.org/0000-0002-3673-4714 Ivanova Olga [email protected] 5 Gorodnicheva Tatiana [email protected] 11 https://orcid.org/0000-0002-7443-6961 Kochetkov Sergey [email protected] 5 Gottikh Marina [email protected] 8 Strumfa Ilze [email protected] 3 https://orcid.org/0000-0002-5659-9679 Ivanov Alexander [email protected] 5 Gordeychuk Ilya [email protected] 1 2 12 https://orcid.org/0000-0001-9382-2254 Isaguliants Maria [email protected] 1 2 3 7 García-Rivas Gerardo 1 NF Gamaleya Research Center of Epidemiology and Microbiology Moscow Russia 2 Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences Moscow Russia chumakovs.ru 3 Department of Pathology Riga Stradins University Riga Latvia rsu.lv 4 Latvian Biomedical Research and Study Centre Riga Latvia lu.lv 5 Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia ras.ru 6 National Medical Research Center for Tuberculosis and Infectious Diseases Moscow Russia 7 Department of Microbiology Tumor and Cell Biology Karolinska Institutet Stockholm Sweden ki.se 8 Chemistry Department and Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia msu.ru 9 Science for Life Laboratory Stockholm University Stockholm Sweden su.se 10 Russian State Center for Quality and Standardization of Veterinary Drugs and Feed (VGNKI) Moscow Russia 11 Evrogen Moscow Russia 12 Sechenov First Moscow State Medical University Moscow Russia mma.ru 2019 2 12 2019 2019 08 05 2019 01 11 2019 05 11 2019 2 12 2019 2019 Copyright © 2019 Ekaterina Bayurova et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist . These properties, particularly, the expression of Twist , correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist . No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression. Ministry of Science and Higher Education of the Russian Federation 075-15-2019-1660 Latvian Science Council LZP-2018/2-0308 EU VACTRAIN Russian Foundation for Basic Research 17-00-00085 17_04_00583 17_54_30002 Publisher Copyright: © 2019 Ekaterina Bayurova et al.HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.publishersversionPeer reviewe

Correction: Zhitkevich et al. HIV-1 Reverse Transcriptase Expression in HPV16-Infected Epidermoid Carcinoma Cells Alters E6 Expression and Cellular Metabolism, and Induces a Hybrid Epithelial/Mesenchymal Cell Phenotype. Viruses 2024, 16, 193.

Author Juris Jansons requested his exclusion from the authors of the original publication [...]

HIV-1 Reverse Transcriptase Expression in HPV16-Infected Epidermoid Carcinoma Cells Alters E6 Expression and Cellular Metabolism, and Induces a Hybrid Epithelial/Mesenchymal Cell Phenotype.

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells

Replenishment of hepatitis B virus cccDNA pool is restricted by baseline expression of host restriction factors in vitro

Funding Information: National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia; [email protected] (A.K.); [email protected] (V.C.) Institute of Immunology, Federal Medical Biological Agency, 115522 Moscow, Russia; [email protected] NF Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia; [email protected] (E.B.); [email protected] (I.G.); [email protected] (M.I.) Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia Sechenov First Moscow State Medical University, 119146 Moscow, Russia; [email protected] Riga Stradins University, LV-1007 Riga, Latvia Karolinska Institutet, SE-171 76 Stockholm, Sweden Izmerov Research Institute of Occupational Health, 105275 Moscow, Russia; [email protected] (I.G.); [email protected] (A.N.) Institute of Medical Virology, University of Giessen, 35392 Giessen, Germany; [email protected] 10 Central Research Institute of Epidemiology, 111123 Moscow, Russia Funding Information: Funding: This work was funded by the Russian Science Foundation grant No. 16-15-10426. D.K. received a scholarship from the President of Russia no. SP1439.2019.4. Publisher Copyright: © 2019 by the authors. Licensee MDPI, Basel, Switzerland.Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. Methods: HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. CeSavell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. Results: Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4-to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized γ-H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression. Conclusion: Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression.publishersversionPeer reviewe

HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape

HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape