Gag Regions Research Articles

Identification of Two Novel HIV-1 Unique Recombinant Forms (CRF01_AE/CRF07_BC) and Genomic Characterization in Tongzhou District of Beijing, China.

Continuous recombination and variation during replication could lead to rapid evolution and genetic diversity of HIV-1. Some studies had identified that it was easy to develop new recombinant strains of HIV-1 among the populations of men who have sex with men (MSM). Surveillance of genetic variants of HIV-1 in key populations was crucial for comprehending the development of regional HIV-1 epidemics. The finding was reported the identification of two new unique recombinant forms (URF 20110561 and 21110743) from individuals infected with HIV-1 in Tongzhou, Beijing in 2020-2022. Sequences of near full-length genome (NFLG) were amplified, then identification of amplification products used phylogenetic analyses. The result showed that CRF01_AE was the main backbone of 20110561 and 21110743. In the gag region of the virus, 20110561 was inserted two fragments from CRF07_BC, while in the pol and tat regions of the virus, 21110743 was inserted four fragments from CRF07_BC. The CRF01_AE parental origin in the genomes of the two URFs was derived from the CRF01_AE Cluster 4. In the phylogenetic tree, the CRF07_BC parental origin of 20110561 clustered with 07BC_N and the CRF07_BC parental origin of 21110743 clustered with 07BC_O. In summary, the prevalence of novel second-generation URFs of HIV-1 was monitored in Tongzhou, Beijing. The emergence of the novel CRF01_AE/CRF07_BC recombination demonstrated that there was a great significance of continuous monitoring of new URFs in MSM populations to prevent and control the spreading of new HIV-1 URFs.

Sensing of an HIV-1-Derived Single-Stranded RNA-Oligonucleotide Induces Arginase 1-Mediated Tolerance.

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV-1-derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV-1-derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases.

A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein.

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

Detection of lymphoproliferative disease virus in Iowa Wild Turkeys (Meleagris gallopavo): Comparison of two sections of the proviral genome.

An accurate diagnostic test is an essential aspect of successfully monitoring and managing wildlife diseases. Lymphoproliferative Disease Virus (LPDV) is an avian retrovirus that was first identified in domestic turkeys in Europe and was first reported in a Wild Turkey (Meleagris gallopavo) in the United States in 2009. It has since been found to be widely distributed throughout North America. The majority of studies have utilized bone marrow and PCR primers targeting a 413-nucleotide sequence of the gag gene of the provirus to detect infection. While prior studies have evaluated the viability of other tissues for LPDV detection (whole blood, spleen, liver, cloacal swabs) none to date have studied differences in detection rates when utilizing different genomic regions of the provirus. This study examined the effectiveness of another section of the provirus, a 335-nucleotide sequence starting in the U3 region of the LTR (Long Terminal Repeat) and extending into the Matrix of the gag region (henceforth LTR), for detecting LPDV. Bone marrow samples from hunter-harvested Wild Turkeys (n = 925) were tested for LPDV with the gag gene and a subset (n = 417) including both those testing positive and those where LPDV was not detected was re-tested with LTR. The positive percent agreement (PPA) was 97.1% (68 of 70 gag positive samples tested positive with LTR) while the negative percent agreement (NPA) was only 68.0% (236 of 347 gag negative samples tested negative with LTR). Cohen's Kappa (κ = 0.402, Z = 10.26, p<0.0001) and the McNemar test (OR = 55.5, p<0.0001) indicated weak agreement between the two gene regions. We found that in Iowa Wild Turkeys use of the LTR region identified LPDV in many samples in which we failed to detect LPDV using the gag region and that LTR may be more appropriate for LPDV surveillance and monitoring. However, neither region of the provirus resulted in perfect detection and additional work is necessary to determine if LTR is more reliable in other geographic regions where LPDV occurs.

Occurrence of Typical Domestic Animal Viruses in Wild Carnivorans: An Emerging Threat to the Conservation of Endangered Species

Wild species are susceptible to several typical domestic animal pathogens, and the increasingly close contact between these groups is a predictive factor for disease exposure. Some viruses are important and old-known, and others are emerging or reemerging for domestic carnivorans and have been identified as threats to the conservation of wild mammals. The purpose of the study was to investigate the occurrence of bocaparvoviruses (BoVs, Parvoviridae family, Parvovirinae subfamily, Bocaparvovirus genus), parvoviruses (Parvoviridae family, Parvovirinae subfamily, Protoparvovirus genus, Protoparvovirus carnivoran1), hepadnaviruses (Hepadnaviridae family), coronaviruses (Coronaviridae family, Orthocoronavirinae subfamily), paramyxoviruses (Paramyxoviridae family) and canine distemper virus (Orthoparamyxovirinae subfamily, Morbillivirus genus, Morbillivirus canis), poxviruses (Poxviridae family), feline herpesvirus (Orthoherpesviridae family, Alphaherpesvirinae subfamily, Varicellovirus genus, Varicellovirus felidalpha1), feline calicivirus (Caliciviridae family, Vesivirus genus, FCV), feline immunodeficiency virus (Retroviridae family, Orthoretrovirinae subfamily, Lentivirus genus, FIV), feline leukemia virus (Retroviridae family, Orthoretrovirinae subfamily, Gammaretrovirus genus, FeLV), and gammaherpesviruses (Orthoherpesviridae family, Gammaherpesvirinae subfamily) in wild carnivorans. A total of 30 biological samples from the families Canidae, Felidae, Mephitidae, Mustelidae, and Procyonidae were evaluated. All animals were victims of vehicular collisions in the state of Mato Grosso do Sul, Brazil. Canine parvovirus (CPV-2) DNA was detected in the spleen of a bush dog (Speothos venaticus), a jaguarundi (Puma yagouaroundi), and a jaguar (Panthera onca), FeLV proviral DNA was found in the spleen of an ocelot (Leopardus pardalis); while CDV RNA was detected in the liver of a jaguarundi. Phylogenetic analysis carried out with the partial sequence of the CPV-2 VP2 gene and the U3 (LTR) gag region of FeLV showed 100% identity with strains obtained from domestic dogs and cats, respectively. The approximation between wild and domestic animals favors the transmission of pathogens, especially between phylogenetically close species, such as members of the Canidae and Felidae families. Identification of the DNA and RNA of potentially fatal viruses such as CPV-2, FeLV, and CDV in four wilds endangered to extinction and understudied species contributes to our understanding of the pathogens circulating in this free-ranging and vulnerable population.

Evaluation of Rapid Molecular Viral Load Monitoring with ABBOTT m-PIMATM HIV-1/2 VL

World Health Organization recommends rapid results of Human Immunodeficiency Virus (HIV) viral load by using Point of Care (POC) testing, delivering faster decision-making to prevent transmission and suppress drug resistance and greater likelihood of starting therapy if the clinic provides care and treatment services. The m-PIMATM HIV-1/2VL cartridge is an RT-PCR POC test that detects HIV-1 and HIV-2 RNA viral load, recognizing the conserved-part 5'-UTR of the HIV genome. The standard, Roche Cobas®4800 system HIV-1, targets gag and LTR regions. This study aimed to determine the correlation, sensitivity, and specificity of Abbott m-PIMATM HIV-1/2VL compared to Roche Cobas®4800 system HIV-1. A cross-sectional study at Dharmais Hospital Laboratory. EDTA plasma obtained from 200 HIV patients, consisted of 80 high-viral-load samples (&gt;1000 copies/mL), 80 low-viral-load samples (&lt;1000 copies/mL), and 40 undetectable-viral-load samples (&lt;14.2 copies/mL). Kappa Cohen was used to measure the categorical correlation. Sensitivity and specificity were calculated with diagnostic test. Dharmais Ethic Committee released ethical clearance No.0132/KEPK/IX/2020. The categorical correlation of Abbott m-PIMATM HIV-1/2 VL in determining the group of high or low viral load samples was 0.948 (very strong correlation). The categorical correlation deciding whether the HIV viral load sample was high, low, or undetectable was 0.714 (strong correlation). The categorical sensitivity of Abbott m-PIMATM HIV-1/2 VL compared to Roche Cobas®4800 system HIV-1 in categorizing HIV viral load was 97.5%, specificity of 97.5%. Abbott m-PIMATM HIV-1/2 VL performance in determining HIV RNA viral load group samples has a strong correlation compared to Roche Cobas®4800 system HIV-1. A larger study is required.

HIV-2 Immature Particle Morphology Provides Insights into Gag Lattice Stability and Virus Maturation

Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we have used the single particle cryo-electron microscopy method to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map at 5.5 Å resolution revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag lattice structures. Cryo-electron tomography provided evidence for nearly complete ordered Gag lattice structures in HIV-2 immature particles. We also solved a 1.98 Å resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10–12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide the first 3D organization of HIV-2 immature Gag lattice and important insights into both HIV Gag lattice stabilization and virus maturation.

Palladium encapsulated mesoporous silica nanoparticles for the rapid detection of analytes.

We designed a simple, inexpensive, and user-friendly assay using mesoporous silica nanoparticles to detect analytes. Highly stable and uniform palladium nanoparticles covered with mesoporous silica (Pd@mSiO2) were generated and characterized extensively using physical methods. Human Serum Albumin (HSA) protein or ssDNA specific to the HIV gag region was capped onto the Pd@mSiO2 electrostatically. This "cap" prevented the Pd(0) inside the mesoporous silica nanoparticles from catalyzing the conversion of non-fluorescent molecules to fluorescent molecules. In the presence of target anti-HSA antibodies or complementary sequence (HIV gag), HSA protein or DNA cap dissociated from the surface of Pd@mSiO2-NH2 through the specific antigen-antibody reaction or DNA hybridization, allowing Pd(0) to convert the non-fluorescent molecules to fluorescent molecules. The limit and range of detection of anti-HSA antibodies were 3.8 nM and 3.8 nM to 133.3 nM, respectively. The limit and range of detection of HIV gag were 1.6 nM and 1.6 nM to 15 nM, respectively. This simple, inexpensive, "add sample and measure" diagnostic assay could potentially be incorporated into point of care diagnostics for low-resource settings.

Phylogenetic Analysis of Small Ruminant Lentiviruses Originating from Naturally Infected Sheep and Goats from Poland Based on the Long Terminal Repeat Sequences.

Previous gag and env sequence studies placed Polish small ruminant lentiviruses (SRLVs) isolated from sheep and goats in subtypes B1, B2, A1, A5, A12, A13, A16-A18, A23, A24 and A27. This study extended the genetic/phylogenetic analysis of previously identified Polish SRLV strains by contributing long terminal repeat (LTR) sequences. A total of 112 samples were analysed. Phylogenetic analyses were carried out on the LTR fragment using the neighbour-joining, maximum likelihood, and unweighted pair group method with arithmetic mean methods. Polish caprine and ovine LTR sequences clustered within group A and grouped in at least 10 clusters (subtypes A1, A5, A12, A13, A16-A18, A23, A24 and A27). Most of the Polish strains (78%) belonged to the same subtype by the indication of the gag, env and LTR genomic regions. Discrepancies in affiliation depending on the particular sequence were observed in 24 (21%) strains, most of which came from mixed-species flocks where more than one SRLV genotype circulated. Sequences of the LTR reflected subtype-specific patterns. Several subtype-specific markers were identified, e.g. a unique substitution of T to A in the fifth position of the TATA box in A17, A27, A20 and B3. This study provides valuable insights into the genetic diversity of SRLV field strains in Poland, their phylogenetic relationships and their position in the recently established SRLV classification. Our results confirmed the existence of the ten subtypes listed and the readier emergence of new SRLV variants in mixed-species flocks.

Viral and Host Factors Regulating HIV-1 Envelope Protein Trafficking and Particle Incorporation.

The HIV-1 envelope glycoprotein (Env) is an essential structural component of the virus, serving as the receptor-binding protein and principal neutralizing determinant. Env trimers are incorporated into developing particles at the plasma membrane of infected cells. Incorporation of HIV-1 Env into particles in T cells and macrophages is regulated by the long Env cytoplasmic tail (CT) and the matrix region of Gag. The CT incorporates motifs that interact with cellular factors involved in endosomal trafficking. Env follows an unusual pathway to arrive at the site of particle assembly, first traversing the secretory pathway to the plasma membrane (PM), then undergoing endocytosis, followed by directed sorting to the site of particle assembly on the PM. Many aspects of Env trafficking remain to be defined, including the sequential events that occur following endocytosis, leading to productive recycling and particle incorporation. This review focuses on the host factors and pathways involved in Env trafficking, and discusses leading models of Env incorporation into particles.

Genetic characterization of varicella-zoster and HIV-1 viruses from the cerebrospinal fluid of a co-infected encephalitic patient, Ghana

BackgroundEncephalitis is a serious disease of the brain characterized by prodromal and specific neurological symptoms. HIV infections offer opportunistic viruses, such as Varicella-zoster virus (VZV), the chance to cause encephalitis in patients. There is a lack of information on the genetic diversity of VZV in Ghana and other parts of Africa which requires sequencing and characterization studies to address. The active evolution of HIV-1 in West Africa also requires continuous surveillance for the emergence of new genetic forms.Case presentationVZV was detected in the CSF sample of an 11-year-old patient presenting with symptoms of encephalitis by real-time PCR diagnostics. To identify possible unknown aetiological pathogens, next-generation sequencing was performed, and revealed an HIV-1 co-infection.Alignments of concatenated HIV-1 genome fragments in the gag, pol, vif, env and nef regions and a near-complete VZV genome were analyzed by Bayesian inference, and phylogenetic trees were generated. The VZV sequence belongs to clade 5 and the HIV-1 sequence is a member of the CRF02_AG predominant circulating recombinant form in Ghana.ConclusionsDiagnostic tests for CSF HIV would be useful where possible in patients presenting with encephalitis due to VZV and other opportunistic viruses in Kumasi to shed light on the role of HIV in encephalitis cases in Ghana. This report reaffirms the role of the CRF02_AG circulating recombinant form in HIV infections in Ghana and also gives a preliminary genetic characterization of VZV in Kumasi, Ghana.

Selective human cells in vitro transduction with porcine endogenous retrovirus (PERV).

Porcine xenograft transplantation raises concerns in humans about the risk of infection with porcine endogenous retroviruses (PERV) as they are an integral part of the pig genome and are therefore very difficult to exclude. In this study, for the first time, arelationship between the provirus genes sequences and released virions from pig cell line and the embedded sequence of this retrovirus in infected human cells was analyzed. PERV infection of human cells HEK-293 and HeLa and detection of PERV in pig PK-15 cells and supernatant were assessed by QPCR or RT-QPCR using primers specific for envA, envB, gag, pol genes and LTR region. Sequence analysis was performed at the DNA level and changes in the amino acid sequence were deduced in silico. Fifty nucleotide substitutions (45 in pol, 3 in gag and one each in envA and envB) were detected and most of these were heterozygous (42), which were present mainly in PK-15 cells. Our results show that sequence of the pol gene and the Pol protein is less conserved compared to the other PERV genes and PERV with some polymorphisms were not released from pig cells or/and do not infect human cells. PERV virions with ahomozygous allele system were released from PK-15 cells, although their sequence replicated on the basis of the heterozygous PERV provirus sequence in PK-15. The newly discovered selective transduction of human cells with PERV will be helpful in studying the characteristics and genetic variability of the retrovirus genes to ensure safe xenotransplantation. Keywords: PERV; porcine endogenous retroviruses; infection; genetic polymorphism; xenotransplantation.

A quantitative analysis of the similarities and differences of the HIV/FIV gag genome

Among viruses, human immunodeficiency virus (HIV) presents the greatest challenge to humans. Here, we retrieved genome sequences from NCBI and were then run through LALIGN bioinformatics software to compute the E value, bit score, Waterman eggert score, and percent identity, which are four important indicators of how similar the sequences are. These 4 values are 3.1 x 10^-9,51.9, 241 and 55.4%.Bases 1600 to 1990 in HIV and bases 800 to 910 in FIV have a higher than normal similarity. This reflects that while the DNA sequences of the gag region of both the HIV and FIV genomes are rather similar, it is unlikely that this similarity is due to random chance; therefore, there are a noticeable number of differences. A better understanding of the level of similarity and differences in the gag region of the genome sequence would facilitate our understanding of structural and cellular behavioral differences between FIV and HIV, and in the long term, it will provide new insights into the differences observed in previous studies or even facilitate the development of an effective HIV treatment.

A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors.

Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5′ leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5–gag interaction and a more stable structure with a U5–AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5′ region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.

NtMYB305a binds to the jasmonate-responsive GAG region of NtPMT1a promoter to regulate nicotine biosynthesis.

MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, which comprises a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30-bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.

Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro.

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat’s immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.

Immunovirological Assessment of HIV-Infected Patients and Phylogenetic Analysis of the Virus in Northeast of Iran

Background: The reliable laboratory tests, co-infection with other tumor viruses, and determining the genetic types are very important for therapy and monitoring of the clinical status of human immunodeficiency virus (HIV)-infected subjects. Objectives: This study evaluated the co-infection of HIV with hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type 1 (HTLV-1), the viral load, the progression of infection, and its correlation with the clinical status. Methods: Twenty HIV-infected cases were assessed for T cell subpopulations, HBV, HCV, and HTLV-1 co-infection, as well as the viral load. For phylogenetic relationships analysis, the HIV-c2-v5 fragment and p17 of gag were amplified, sequenced, and then clustered using phylogenetic analysis by MEGA software with maximum-likelihood. Results: The quantity of HIV viral load by qRT-PCR (TaqMan) and Cobas-Amplicor monitor test had a very strong correlation (R = 0.881, P &lt; 0.0001). A significant negative correlation was also found between CD4+ cell count and Cobas-Amplicor (R = -0.41, P = 0.06). A significant negative correlation was also found between CD4+ cell count and Cobas-Amplicor (R = -0.41, P = 0.06) with HIV monitor test results (R = -0.41, P = 0.06). The phylogenetic analysis for p17 regions in gag and c2-v5 in env genes showed that all subjects had AD genotype. The co-infection of the HIV subjects with HBV, HCV, and HTLV-1 was 75%, 75%, and 15%, respectively. A direct correlation was observed between CD8+ and HIV-HTLV-1 co-infection. Conclusions: The results showed that HIV CRF35-AD, (M group) is more frequent in the northeast of Iran, and both real-time quantification methods were reliable for monitoring the HIV-1 viral load. In addition, the transmission rate of HTLV-1 is lower than HBV and HCV among drug abusers.

HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

Many lentiviral vectors used for gene therapy are derived from HIV-1. An optimal vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient’s genome. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. Because the splicing profile for lentiviral vectors is poorly characterized, we used long-read sequencing to determine canonical and cryptic splice site usage. Deleting specific regions of env sequence reduced the number of splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining a large deletion in gag with repositioning the Rev-response element downstream of the 3’ R to prevent its reverse transcription showed that 1201 nucleotides of HIV-1 sequence can be removed from the integrated vector genome without substantially compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve safety and transfer less viral sequence into a patient’s DNA.

The HIV 5' Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity.

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.

Infection by Diverse HIV-1 Subtypes Leads to Different Elevations in HERV-K Transcriptional Levels in Human T Cell Lines.

Human endogenous retroviruses (HERVs) make up ~8% of the human genome, and for millions of years, they have been subject to strict biological regulation. Many HERVs do not participate in normal physiological activities in the body. However, in some pathological conditions, they can be abnormally activated. For example, HIV infection can cause abnormal activation of HERVs, and under different infection conditions, HERV expression may be different. We observed significant differences in HERV-K transcription levels among HIV-1 subtype-infected individuals. The transcriptional levels in the HERV-K gag region were significantly increased in HIV-1 B subtype-infected patients, while the transcriptional levels in the HERV-K pol region were significantly increased in CRF01_AE and CRF07_BC subtype-infected patients. In vitro, the transcriptional levels of HEVR-K were increased 5-fold and 15-fold in MT2 cells transfected with two different HIV-1 strains (B and CRF01_AE, respectively). However, there was no significant difference in transcriptional levels among regions of HERV-K. When MT2 cells were infected with different subtypes of HIV-1 Tat proteins (B, CRF01_AE), which is constructed by lentiviruses, and the transcription levels of HERV-K were increased 4-fold and 2-fold, respectively. Thus, different subtypes of HIV-1 have different effects on HERV-K transcription levels, which may be caused by many factors, not only Tat protein.