This study aimed to evaluate by means of Nested Polymerase Chain Reaction (nPCR), co-cultivation and sequencing, with genetic comparison between strains (mother/newborn), the occurrence of vertical transmission of Small Ruminant Lentiviruses (SRLV) from naturally occurring nannies infected for their offspring. For the detection of SRLV seropositive progenitors, blood was collected from 42 nannies in the final third of gestation in tubes with and without anticoagulant. The diagnostic tests used were Western Blot (WB) and nPCR. During the period of birth, the same blood collection procedure was performed on 73 newborns at zero hours of birth, with the same diagnostic tests. Seventeen blood samples from seven-day-old kids, proven positive for SRLV by nPCR, chosen at random, were subjected to coculture in goat synovial membrane (GSM) cells for 105 days. The pro-viral DNA extracted from the cell supernatant from the coculture was subjected to nPCR. For DNA sequencing from the nPCR products, nine positive samples were chosen at random, four nannies with their respective offspring, also positive. Each sample was performed in triplicate, thus generating 27 nPCR products of which only 19 were suitable for analysis. Among the 42 pregnant goats, in 50% (21/42) pro-viral DNA was detected by nPCR, while in the WB, only 7.14% (3/42) presented antibodies against SRLV. Regarding neonates, of the 73 kids, 34 (46.57%) were positive for the virus, using the nPCR technique, while in the serological test (WB), three positive animals (4.10%) were observed. The coculture of the 17 samples with a positive result in the nPCR was confirmed in viral isolation by amplification of the SRLV pro-viral DNA. When aligned, the pro-viral DNA sequences (nannies and their respective offspring) presented homology in relation to the standard strain CAEV Co. It was concluded that the transmission of SRLV through intrauterine route was potentially the source of infection in the newborn goats.

/pdf/vertical-transmissibility-of-small-ruminant-lentivirus-36p12mnmov.pdf

Vertical transmissibility of small ruminant lentivirus.

HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-β conformation, which interacts with glycosaminoglycans (GAGs) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-β variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s9 loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues: R18, R35, and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (∼350 A 2 ) positively charged surface in the all-β XCL1 conformation, whereas they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors. IMPORTANCE The host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of noncytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8 + T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-β fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-β XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.

Structural Determinants for the Selective Anti-HIV-1 Activity of the All-β Alternative Conformer of XCL1

The HIV-1 Env spike is the main protein complex that facilitates HIV-1 entry into CD4+ host cells. HIV-1 entry is a multistep process that is not yet completely understood. This process involves several protein-protein interactions between HIV-1 Env and a variety of host cell receptors along with many conformational changes within the spike. HIV-1 Env developed due to high mutation rates and plasticity escape strategies from immense immune pressure and entry inhibitors. We applied a coevolution and residue-residue contact detecting method to identify coevolution patterns within HIV-1 Env protein sequences representing all group M subtypes. We identified 424 coevolving residue pairs within HIV-1 Env. The majority of predicted pairs are residue-residue contacts and are proximal in 3D structure. Furthermore, many of the detected pairs have functional implications due to contributions in either CD4 or coreceptor binding, or variable loop, gp120-gp41, and interdomain interactions. This study provides a new dimension of information in HIV research. The identified residue couplings may not only be important in assisting gp120 and gp41 coordinate structure prediction, but also in designing new and effective entry inhibitors that incorporate mutation patterns of HIV-1 Env.

/pdf/coevolution-analysis-of-hiv-1-envelope-glycoprotein-complex-qsrm2t1dif.pdf

Coevolution Analysis of HIV-1 Envelope Glycoprotein Complex.

Mother-to-child transmission (MTCT) of HIV offers a good opportunity to study the dynamics of early viral evolution in the host environment to which the virus has partially adapted. Such studies would throw light on the unique features of the infecting viruses, which will subsequently help to design preventive or therapeutic measures against the newly infecting and evolving strains of HIV. Therefore, we undertook a study to determine the genetic divergence of proviral envelope sequences from the HIV-infected infants (<2 years). Detailed analysis revealed unique features of potential N-linked glycosylation sites (PNGS) and their frequency of occurrence that built on the difference in length of the V1V2 region of the envelope sequences. Surprisingly, frequency of PNGS in the V5 region was found to revert rapidly, in about 75% of the sequences, which could surmise a fitness disadvantage in the variant forms. Further, a stable net charge was observed in the V2 and V3 regions prompting us to speculate...

Molecular Characteristics of the Envelope of Vertically Transmitted HIV-1 Strains from Infants with HIV Infection.

https://link.springer.com/content/pdf/10.1007%2Fs12250-016-3725-5.pdf

The V1 region of gp120 is preferentially selected during SIV/HIV transmission and is indispensable for envelope function and virus infection

The importance of the fourth variable (V4) region of the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein (Env) in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS). In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS), greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain) resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.

/pdf/critical-amino-acids-within-the-human-immunodeficiency-virus-wa2lk9t8ai.pdf

Critical Amino Acids within the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein V4 N- and C-Terminals Contribute to Virus Entry

The invention discloses an expression method of a high secretory tripolymer protein. The expression method comprises the following steps of: (1) connecting a tPA signal peptide gene sequence, a target protein gene and a protein trimerization modif gene sequence together; wherein N tail end of the target protein is connected with the tPA signal peptide, and C tail end of the target protein is connected with N tail end of the protein timerization modif protein; (2) cloning a fused gene to an eukaryotic expression vector to obtain a fused protein eukaryotic expression vector; and (3) finally expressing the protein in vivo or in vitro. By importing a signal peptide sequence of a tissue-type plasminogen activator tPA to an amino terminal of the target protein and importing a trimerization protein modif MTQ to a carboxyl terminal of the target protein, the invention realizes that the target protein has a high level secretion and a trimerization expression in an eukaryotic cell to obtain a functional protein. The invention can be widely applied to viruses which are naturally in the form of tripolymer and the high level secretory expressions of other proteins in the eukaryotic cells.

Expression method of a high secretory tripolymer protein

The invention discloses an expression method for high-secretory tripolymer protein. The expression method comprises the following steps of: (1) connecting a tPA signal peptide gene sequence, a target protein gene sequence and a protein trimerization modif gene sequence together, wherein the N tail end of the target protein is connected with the tPA signal peptide, and the C tail end of the targetprotein is connected with the N tail end of the protein trimerization modif protein; (2) cloning a fusion gene to a prokaryotic or eukaryotic expression vector to obtain a fusion protein prokaryotic or eukaryotic expression vector; and (3) expressing the fusion protein in vivo or in vitro. The method realizes high-level secretion and trimerization expression of the target protein in prokaryotic or eukaryotic cells to obtain functional protein by introducing a signal peptide sequence of an organized plasminogen activator tPA at the amino terminal of the target protein and introducing a trimerization protein modif MTI at the carboxyl end of the target protein. The method can be widely applied to viruses which are naturally in the form of tripolymer and the high-level secretory expressions of other proteins in the prokaryotic or eukaryotic cells.

Expression method for protein trimerization modif-based high-secretory tripolymer protein

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Critical Amino Acids within the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein V4 N- and C-Terminals Contribute to Virus Entry

Expression method of a high secretory tripolymer protein

Expression method for protein trimerization modif-based high-secretory tripolymer protein