Retrovirology最新文献

Detecting and measuring HIV reservoirs, neutralizing antibodies, and restriction factors are important for HIV cure research and the development of new therapeutics and vaccines. Here we describe the development and validation of several HIV Rev-dependent indicator cell lines for these purposes. These reporter cells derive from different T-lymphoblast cell lines, including Molt4-CCR5, SupT1-CCR5, CEM-SS, A3R5, and from the adherent TZM cell platform based on HeLa clone JC53. These cells express CD4, CXCR4, and various levels of CCR5. We compared these cell lines for responsiveness to both X4 and R5-tropic viruses, and confirmed that reporter expression in these cells is not affected by stimulation from mitogens but is responsive to HIV Tat and Rev, reducing non-specific reporter induction from the leaky LTR promoter. To validate the sensitivity of the Rev-dependent reporter cell systems, we conducted a viral dilution assay with three primary HIV-1 clade C swarms from an adult in Malawi. We also validated the systems for quantifying antibody neutralization and screening restriction factors; these systems are also sensitive for viral outgrowth assays for quantifying viral reservoirs in clinical and basic research settings. Given that the systems can measure HIV accurately in complex environments with mitogens or other substances, they can be used for versatile applications, such as quantifying latent reservoirs, testing inhibitory compounds, conducting neutralizing antibody assays, and identifying new restriction factors.

Background: Breast cancer is the most commonly diagnosed cancer globally. The potential role of oncogenic viruses, particularly Human Betaretrovirus (HBRV, formerly MMTV-LV/HMTV), in the pathogenesis of breast cancer has been a subject of research for decades. However, studies investigating this association have produced conflicting results. This systematic review and meta-analysis aim to assess the prevalence of HBRV in breast cancer cases and evaluate its potential association with breast cancer.

Methods: A systematic literature search was conducted in MEDLINE, Web of Science, Scopus, and EMBASE following PRISMA guidelines. Studies assessing HBRV prevalence in breast cancer patients and case-control studies investigating its association with breast cancer risk were included. The Newcastle-Ottawa Scale (NOS) was used to evaluate study quality, and meta-analysis was performed using RevMan 5.1. Heterogeneity was assessed using the I² statistic, and subgroup analyses were conducted based on detection methods, sample types, and geographic regions.

Results: The literature search identified a total of 45 studies that were deemed suitable for inclusion in the systematic review. 26 studies were used in the subsequent meta-analysis. The initial meta-analysis revealed a significant association between HBRV and breast cancer (OR = 4.92, 95% CI: 4.00-6.04, p < 0.00001) but exhibited high heterogeneity (I² = 82%). After excluding an outlier, heterogeneity was significantly reduced (I² = 22%), with a revised OR of 11.95 (95% CI: 8.78-16.25, p < 0.00001 ). Subgroup analysis demonstrated variation in detection methods, with Nested PCR (OR = 19.15) and Frozen tissue samples (OR = 18.00) showing the strongest associations. Geographic analysis indicated the highest odds in North America (OR = 24.75), followed by Europe (OR = 15.02).

Conclusion: This meta-analysis suggests strong epidemiological evidence supporting an association between HBRV infection and human breast cancer, and is consistent with a possible etiological role. However, variability in study methodologies and geographic differences warrant further investigation through standardized, large-scale studies to confirm these findings and explore potential mechanisms of viral oncogenesis in breast cancer.

Although HPV infection is obligatory for almost all cases of cervical cancer (CC), other risk factors can promote the progression of cervical cancer. In this context, the expression of human endogenous retroviruses (HERVs) in the development of CC has been investigated. In this study, the expression status of HERV-E env transcripts was analyzed in 111 cervical biopsies, including 35 cervical cancer samples, 20 precancerous lesions, and 56 normal samples. Real-time PCR with specific primers was used to quantify the relative expression of HERV-E env, HPV 16 and 18 E6/E7 genes, and GAPDH as a normalization control. Our results indicated an increase in the expression of HERV-E env, and the difference was statistically significant in the cancer group compared to the precancerous group (1.5-fold change) (P = 0.031). In HPV 16 or 18-infected patients, a higher mean value of HERV-E env mRNA was also found in the cancer group than in the precancerous group. ROC curve analysis showed a significant difference in env expression between precancerous and cancerous lesions in all patients analyzed (P = 0.015) and in a group of patients infected with HPV 16 or 18 genotypes (P = 0.023). In addition, there was a positive correlation between the higher expression of HERV-E env mRNA with E7 (R = 0.34, P = 0.016) and age (R = 0.35, p = 0.016) in HPV 16-infected patients. In conclusion, our study found a possible association between HERV-E env expression and cervical cancer, as HERV-E is actively transcribed during the progression of cervical lesions. Future studies on the potential interaction of HERV-E env with HPV 16 E7 oncoprotein are likely to elucidate common signaling pathways in the progression of cervical cancer and other HPV-related malignancies.

Background: Variation in the level of cell-associated HIV-1 RNA is an important parameter followed in clinical trials focusing on HIV-1 infection, latency or reactivation. In addition to sense products, HIV-1 also expresses antisense products that can modulate HIV-1 replication, either positively through the antisense protein ASP or negatively through repressive noncoding antisense RNAs. Therefore, quantification of both sense and antisense HIV-1 products could provide key information for monitoring the dynamics of viral replication in vivo. While the ASP protein is difficult to detect even in vitro, antisense RNAs can be detected both in vitro and in vivo. The aim of this study was therefore to establish protocols for the specific quantification of sense and antisense transcription that can be applied in clinical studies. To this end, we developed strand-specific RT-PCR protocols allowing us to quantify individually sense and antisense RNAs in PLWH with B and non-B viruses.

Results: We show that the two RTqPCR protocols can quantify standard HIV-1 sequences with good analytical parameters. We also demonstrate the strand specificity of the two protocols by showing that RNAs of the other orientation do not contaminate RNAs of one orientation during the PCR step and that the sense and antisense RT-qPCR protocols detect distinct populations of HIV-1 RNAs. We then compared the sensitivity of RT-qPCR and RT-ddPCR to quantify HIV-1 cell-associated RNAs and found that RT-ddPCR results in lower inter-sample variation or higher levels of detection than RT-qPCR. Finally, we show that the RT-ddPCR protocols efficiently quantify cell-associated HIV-1 sense and antisense RNAs not only in HIV-1-infected primary CD4 + T cells but also in spleen and blood samples from untreated HIV-1-infected individuals.

Conclusions: This study demonstrates that HIV-1 antisense RNAs are expressed in spleen and blood of untreated HIV-1-infected individuals, of at least B and CRF02 subtypes, and that the level of antisense transcription can be significant and even predominant, as compared to sense transcription. These data, along with the protocols we described, will enable a more thorough analysis of HIV-1 sense and antisense expression dynamics in vivo, which could pave the way for novel strategies to control HIV-1 infection.

Background: The evolutionary history of retroviruses and their impact on vertebrate evolution remains poorly understood, particularly in non-mammalian hosts. In this study, we explore retroviruses associated with Amphibia through analysis of 169 RNA sequencing datasets from 102 amphibian species. Using a BLAST-based approach, we identified retroviral transcripts from assembled transcriptomes and phylogenetically characterise both their pol and env regions to elucidate their evolutionary history.

Results: We identified the transcription of 18 novel and two previously described retroviruses with closest relatives in gammaretrovirus, epsilonretrovirus, betaretrovirus and spumaretrovirinae. Despite their differing pol phylogenies, we found that all amphibian retroviruses belong to the gamma-type envelope group (GTE). This suggests a common selection pressure for amphibian retroviruses to retain GTEs. Within these GTEs we also observed a new clade of alpharetrovirus-like envelopes in amphibians which form a sister clade to avian alpharetrovirus envelopes. Furthermore, we observe correlations between amphibian taxonomical order and retroviral diversity, with Gymnophiona (caecilians) harbouring the widest diversity of retroviruses whilst Anura (frogs and toads) harbour the fewest. Through mapping these transcribed retroviruses to their respective genomes (seven available) supplemented with observing ORF intactness, we determined that 14 of the 20 retroviruses are likely endogenous in origin yet are still transcribed in many amphibian tissues. These amphibian endogenous retroviruses (ERVs) have high genomic copy numbers: most (5/7) ERVs investigated have > 100 copies, and one of which has 9,219 integrations within the Ichthyophis bannanicus caecilian genome. This high retroviral load in amphibian genomes may suggest that these retroviruses have low pathogenicity, or may reflect a lack of transposon control mechanisms in amphibian cells.

Conclusions: Through the characterisation of metatranscriptomic and genomic data from retroviruses in this study, we provide insights into their evolution in amphibians and exemplify the diversity of Retroviridae in vertebrate genomes. The identification of novel retroviral clades, widespread transcription of endogenous retroviruses in amphibians and abundance of ERV copies suggests that Retroviridae have played a significant role in amphibian evolution.

Background: Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4⁺ T lymphocytes and causes both malignant and inflammatory diseases: the aggressive malignancy known as Adult T-cell leukemia/lymphoma (ATL) and several chronic inflammatory syndromes. HTLV-1 infection is established by integration of proviral DNA (~9000 bp) into the host genome. In HTLV-1, two viral genes, tax and HBZ, play critical roles in viral transcription and promotion of T-cell proliferation, respectively. The present study was undertaken to test the hypothesis that the higher-order structure of proviral chromatin regulates its transcription on both the plus strand and the minus strand.

Results: ATAC-seq analysis identified an open chromatin region in the pol gene of proviral DNA, which we name IPOR, in many ATL cases. Using reporter assays, it was found that the sequence of IPOR suppresses the transcription of the plus strand and activates that of the minus strand. Binding motifs of Eomes and TEAD proteins were predicted in this region, and we confirmed recruitment of the transcription factors to their respective motifs by ChIP-qPCR. A mutant of IPOR which cannot bind the transcription factors weakened the transcriptional activating effects compared with the wild type, suggesting that the IPOR and those transcription factors suppress the 5' long terminal repeat (LTR) but activate the 3'LTR. In addition, a mutant HTLV-1 molecular clone, which possesses the IPOR mutant, produced a higher titer of virus than the wild type. RNA-seq analysis of HTLV-1-infected cell lines, in which Tax expression can be traced after induction, revealed that EOMES expression decreases during the tax transcriptional burst and resumes following termination of the burst. These findings suggested that the expression dynamics of Eomes affect the transient expression of Tax.

Conclusions: The IPOR sequence appears to regulate the transcription from both LTRs, suppressing the 5'LTR but activating the 3'LTR. Recruitment of Eomes to the IPOR is likely to influence the expression of Tax and HBZ. Since both viral genes are involved in diverse mechanisms for viral replication, cellular proliferation, and immune regulation, the IPOR may play a role in fine-tuning the modes of viral persistence in vivo.

Over the past two decades, the global HIV/AIDS pandemic has emerged as one of the most pressing health concerns worldwide. In low-income countries, where resources are scarce, both the prevalence of HIV and the associated mortality rates have been steadily increasing. According to the national AIDS control program, approximately 74,619 individuals in Pakistan are living with HIV/AIDS, with cases distributed across various provinces and autonomous territories. The country's low literacy rate poses a significant barrier to understanding preventive measures, thereby facilitating the uncontrolled transmission of HIV through sexual intercourse, blood transfusions, and the use of contaminated medical equipment. Vulnerable populations include persons engaged in sex work, transgender individuals, males who have sex with men, and those who inject drugs. The movement and relocation of people, particularly in Karachi, are significant factors in the high prevalence of HIV. In Pakistan, where 20% of drug users are HIV positive the HIV positive population and is still spreading among injection drug users (IDUs). Male sex workers and transgender people who engage in sexual interactions with intravenous drug users are experiencing new outbreaks in some locations. HIV risk behaviors are influenced by limited awareness, social stigma, and inadequate harm reduction programs. The HIV/AIDS epidemic is spreading rapidly throughout Pakistan. This review article aims to identify the various factors that are involved in HIV epidemiology in Pakistan.

Background: In the early phase of HIV infection, as studied in vitro, high levels of unintegrated (both linear and circular) and integrated (provirus) forms of viral DNA are seen, and cells produce high levels of virus. In time, the level of unintegrated DNA declines, followed by a progressive decline in virus expression. Extensive studies of the proviral landscape in people living with HIV (PLWH) on antiretroviral therapy (ART) show that only about 2% of proviruses are intact; the remainder are characterized as defective and contain numerous deletions of proviral DNA segments and hypermutations. In the current study, we investigated the decline of viral expression in infected T cells in search of mechanisms involved in proviral inactivation.

Results: We derived clonal lines from Jurkat cells infected with HIV MN and monitored them for viral expression over time in culture. In a subset of clones that displayed a decline in expression, we found provirus containing large deletions and the integration of a retrotranscribed molecule of tRNA^Gly adjacent to the 3'-end of the proviral DNA. We provide evidence linking the proviral deletions to the insertion of a reverse transcribed tRNA^Gly molecule and propose a mechanism for its self-primed reverse transcription.

Conclusions: Large deletions of proviral DNA have been reported in PLWH on ART and attributed to errors that occurred in the synthesis of the minus strand during the reverse transcription of the viral genome. Our results support an additional mechanism for proviral deletions, mediated by tRNA^Gly, in the inactivation of the provirus.

Background: Among the many CRFs, CRF55_01B was the first CRF01_AE and subtype B recombinant strain identified around 2013 among men who have sex with men (MSM) in Shenzhen, China. With rapid spreading throughout the country, CRF55_01B has attracted much attention in recent years. This study aimed to analyze its prevalence of drug resistance and transmission characteristics in people living with HIV/AIDS (PLWHA) in Henan province, China so as to pay particular attention to this group of individuals to reduce the incidence of drug resistance.

Results: Two hundred and forty-five CRF55_01B-infected individuals, including 141 treatment-naïve and 104 treatment-experienced individuals, were enrolled. In treatment-naïve individuals, 6.38% (9/141) of them harboured NRTI DRMs and 19.15% (27/141) of them harboured NNRTI DRMs except V179E/D. In treatment-experienced individuals, 2.00% (2/100) harboured INSTI DRMs, 82.69% (86/104) of them harboured NRTI DRMs, and 88.46% (92/104) of them harboured NNRTI DRMs except V179E/D. The overall prevalence of ADR was 89.42% (93/104), while the prevalence of PDR was 19.86% (28/141). A total of 23 transmission clusters, accounting for 37.55% (92/245) of the total sequences, were identified. The clusters ranged in size from 2 to 19, and 15 (65.22%) had 3 or more sequences.

Conclusions: High prevalence of DRMs and drug resistance were observed in CRF55_01B in both treatment-naïve and treatment-experienced individuals, particular attention should be paid to this group of individuals to reduce the incidence of drug resistance.