Interdomain Linker of HIV/SIV Gag-CA

Gag proteins underlie retroviral replication by fulfilling numerous functional roles at various stages during viral life cycle. Out of the four mature proteins, Gag-capsid (CA) is a major component of viral particles, and has been most well studied biogenetically, biochemically and structurally. Gag-CA is composed of two structured domains, and also of a short stretch of disordered and flexible interdomain linker. While the two domains, namely, N-terminal and C-terminal domains (NTD and CTD), have been the central target for Gag research, the linker region connecting the two has been poorly studied. We recently have performed systemic mutational analyses on the Gag-CA linker region of HIV-1 by various experimental and in silico systems. In total, we have demonstrated that the linker region acts as a cis-modulator to optimize the Gag-related viral replication process. We also have noted, during the course of conducting the research project, that HIV-1 and SIVmac, belonging to distinct primate lentiviral lineages, share a similarly biologically active linker region with each other. In this brief article, we summarize and report the results obtained by mutational studies that are relevant to the functional significance of the interdomain linker of HIV/SIV Gag-CA. Based on this investigation, we discuss about the future directions of the research in this line

Complete Genome Sequences of Human Immunodeficiency Type 1 Viruses Genetically Engineered To Be Tropic for Rhesus Macaques

We have constructed two human immunodeficiency type 1 (HIV-1) derivatives, CXCR4 tropic and CCR5 tropic, that replicate in rhesus macaques. They are genetically engineered to be resistant to macaque restriction factors against HIV-1, including TRIM5α, APOBEC3, and tetherin proteins. The two HIV-1 variants described here are fundamental clones aiming for rhesus infection studies of HIV-1

Nucleotide Variations Affect vif/Vif Expression

Vif is required for HIV-1 replication in natural target cells by counteracting host restriction factors, APOBEC3 (A3) proteins. We recently demonstrated that Vif expression level can be changed by naturally occurring single-nucleotide variations within SA1D2prox of the HIV-1 genome. We also found that levels for vif/vpr mRNAs are inversely correlated. While amino acid sequence per se is critical for functionality, Vif expression level modulated by signal sequences in its coding region is likely to be important as well. There are two splicing sites in the region involved in vpr expression. To reveal possible fluctuations of Vif-expression level, we examined SA1D2prox and vif gene by chimeric approaches using HIV-1 subtypes B and C with distinct anti-A3 activity. In this report, recombinant clones in subtype B backbone carrying chimeric sequences with respect to SA1D2prox/vif and those within the vif-coding region were generated. Of these, clones containing vif-coding sequence of subtype C, especially its 3′ region, expressed vif/Vif at a decreased level but did at an increased level for vpr/Vpr. Clones with reduced vif/Vif level grew similarly or slightly better than a parental clone in weakly A3G-positive cells but more poorly in highly A3G-expressing cells. Three clones with this property were also tested for their A3-degrading activity. One of the clones appeared to have some defect in addition to the poor ability to express vif/Vif. Taken all together, our results show that natural variations in the SA1D2prox and vif-coding region can change the Vif-expression level and affect the HIV-1 replication potential

Review of Mammarenavirus Biology and Replication

The family Arenaviridae is divided into three genera: Mammarenavirus, Reptarenavirus, and Hartmanivirus. The Mammarenaviruses contain viruses responsible for causing human hemorrhagic fever diseases including New World viruses Junin, Machupo, Guanarito, Sabia, and Chapare virus and Old World viruses Lassa, and Lujo virus. These two groups of arenaviruses share the same genome organization composed of two ambisense RNA segments. These segments contain four open reading frames that encode for four proteins: the nucleoprotein, glycoprotein precursor, L protein, and Z. Despite their genome similarities, these groups exhibit marked differences in their replication life cycles. This includes differences in attachment, entry, and immune evasion. By understanding the intricacy of replication in each of these viral species we can work to develop counter measures against human diseases. This includes the development of vaccines and antivirals for these emerging viral threats. Currently only the vaccine against Junin virus, Candid#1, is in use as well as Ribavirin for treatment of Lassa Fever. In addition, small molecule inhibitors can be developed to target various aspects of the virus life cycle. In these ways an understanding of the arenavirus replication cycle can be used to alleviate the mortality and morbidity of these infections worldwide

Search for Molecules against HIV-CA

Varieties of in vitro systems have been used to study biochemical properties of human immunodeficiency virus Gag-capsid protein (HIV Gag-CA). Recently, we have comparatively characterized HIV-1 and HIV-2 Gag-CA proteins using such technology, and have demonstrated that the NaCl-initiated CA-polymerization in vitro and the stability of CA N-terminal domain as judged by differential scanning fluorimetry (DSF) are inversely correlated. In this study, we found that ZnCl2 works as a competent initiator of the in vitro HIV-1 CA-polymerization at much lower concentrations than those of NaCl frequently used for the polymerization initiation. We also showed by DSF assays that ZnCl2 highly destabilize HIV-1 CA. Furthermore, PF74, a well-known inducer of premature HIV-1 uncoating in infected cells, was demonstrated to unusually promote the HIV-1 CA-disassembly in the presence of ZnCl2 as revealed by DSF assays. Taken together, we conclude that the DSF method may be useful as an efficient monitoring system to screen anti-HIV-1 CA molecules

Coronavirus Diversification

Human coronaviruses (HCoVs) are of zoonotic origins, and seven distinct HCoVs are currently known to infect humans. While the four seasonal HCoVs appear to be mildly pathogenic and circulate among human populations, the other three designated SARS-CoV, MERS-CoV, and SARS-CoV-2 can cause severe diseases in some cases. The newly identified SARS-CoV-2, a causative virus of COVID-19 that can be deadly, is now spreading worldwide much more efficiently than the other two pathogenic viruses. Despite evident differences in these properties, all HCoVs commonly have an exceptionally large genomic RNA with a rather peculiar gene organization and have the potential to readily alter their biological properties. CoVs are characterized by their biological diversifications, high recombination, and efficient adaptive evolution. We are particularly concerned about the high replication and transmission nature of SARS-CoV-2, which may lead to the emergence of more transmissible and/or pathogenic viruses than ever before. Furthermore, novel variant viruses may appear at any time from the CoV pools actively circulating or persistently being maintained in the animal reservoirs, and from the CoVs in infected human individuals. In this review, we describe knowns of the CoVs and then mention their unknowns to clarify the major issues to be addressed. Genome organizations and sequences of numerous CoVs have been determined, and the viruses are presently classified into separate phylogenetic groups. Functional roles in the viral replication cycle in vitro of non-structural and structural proteins are also quite well understood or suggested. In contrast, those in the in vitro and in vivo replication for various accessory proteins encoded by the variable 3' one-third portion of the CoV genome mostly remain to be determined. Importantly, the genomic sequences/structures closely linked to the high CoV recombination are poorly investigated and elucidated. Also, determinants for adaptation and pathogenicity have not been systematically investigated. We summarize here these research situations. Among conceivable projects, we are especially interested in the underlying molecular mechanism by which the observed CoV diversification is generated. Finally, as virologists, we discuss how we handle the present difficulties and propose possible research directions in the medium or long term

Appearance of renal hemorrhage in adult mice after inoculation of patient-derived hantavirus

Background: Hemorrhagic fever with renal syndrome (HFRS) caused by hantavirus infection is characterized by fever, renal dysfunction and hemorrhage. An animal model mimicking symptoms of HFRS remains to be established. In this study, we evaluated the pathogenicity of an HFRS patient-derived Hantaan virus (HTNV) in adult mice. Methods: Five clones of HTNV strain KHF 83-61 BL (KHFV) that was derived from blood of an HFRS patient were obtained by plaque cloning. The pathogenicity of the virus clones was evaluated by using 6-week-old female BALB/c mice. Sequence analysis of the viral genome was performed by conventional methods. Results: All of the mice intravenously inoculated with KHFV clone (cl)-1, -2, -3 and -5 showed signs of disease such as transient body weight loss, ruffled fur, reduced activity and remarkably prominent hemorrhage in the renal medulla at 6 to 9 days post-inoculation (dpi) and then recovered. In contrast, mice intravenously inoculated with KHFV cl-4 did not show any signs of disease. We selected KHFV cl-5 and cl-4 as representative of high-pathogenic and low-pathogenic clones, respectively. Quantities of viral RNA in kidneys of KHFV cl-5-infected mice were larger than those in KHFV cl-4-infected mice at any time point examined (3, 6, 9 and 12 dpi). The quantities of viral RNA of KHFV cl-5 and cl-4 peaked at 3 dpi, which was before the onset of disease. Sequence analysis revealed that the amino acid at position 417 in the glycoprotein Gn was the sole difference in viral proteins between KHFV cl-5 and cl-4. The result suggests that amino acid at position 417 in Gn is related to the difference in pathogenicity between KHFV cl-5 and cl-4. When the inoculum of KHFV cl-5 was pretreated with a neutralizing antibody against HTNV strain 76-118, which belongs to the same serotype as KHFV clones, mice did not show any signs of disease, confirming that the disease was caused by KHFV infection. Conclusion: We found that an HFRS patient-derived HTNV caused renal hemorrhage in adult mice. We anticipate that this infection model will be a valuable tool for understanding the pathogenesis of HFRS

HIV-1 Derivatives in Rhesus Macaques

A major issue for present HIV-1 research is to establish model systems that reflect or mimic viral replication and pathogenesis actually observed in infected humans. To this end, various strategies using macaques as infection targets have long been pursued. In particular, experimental infections of rhesus macaques by HIV-1 derivatives have been believed to be best suited, if practicable, for studies on interaction of HIV-1 and humans under various circumstances. Recently, through in vitro genetic manipulations and viral cell-adaptations, we have successfully generated a series of HIV-1 derivatives with CXCR4-tropism or CCR5-tropism that grow in macaque cells to various degrees. Of these viruses, those with best replicative potentials can grow comparably with a pathogenic SIVmac in macaque cells by counteracting major restriction factors TRIM5, APOBEC3, and tetherin proteins. In this study, rhesus macaques were challenged with CXCR4-tropic (MN4/LSDQgtu) or CCR5-tropic (gtu + A4CI1) virus. The two viruses were found to productively infect rhesus macaques, being rhesus macaque-tropic HIV-1 (HIV-1rmt). However, plasma viral RNA was reduced to be an undetectable level in infected macaques at 5–6 weeks post-infection and thereafter. While replicated similarly well in rhesus peripheral blood mononuclear cells, MN4/LSDQgtu grew much better than gtu + A4CI1 in the animals. To the best of our knowledge, this is the first report demonstrating that HIV-1 derivatives (variants) grow in rhesus macaques. These viruses certainly constitute firm bases for generating HIV-1rmt clones pathogenic for rhesus monkeys, albeit they grow more poorly than pathogenic SIVmac and SHIV clones reported to date

Capsid Proteins of HIV-1/HIV-2

Timely disassembly of viral core composed of self-assembled capsid (CA) in infected host cells is crucial for retroviral replication. Extensive in vitro studies to date on the self-assembly/disassembly mechanism of human immunodeficiency virus type 1 (HIV-1) CA have revealed its core structure and amino acid residues essential for CA–CA intermolecular interaction. However, little is known about in vitro properties of HIV-2 CA. In this study, we comparatively analyzed the polymerization properties of bacterially expressed HIV-1 and HIV-2 CA proteins. Interestingly, a much higher concentration of NaCl was required for HIV-2 CA to self-assemble than that for HIV-1 CA, but once the polymerization started, the reaction proceeded more rapidly than that observed for HIV-1 CA. Analysis of a chimeric protein revealed that N-terminal domain (NTD) is responsible for this unique property of HIV-2 CA. To further study the molecular basis for different in vitro properties of HIV-1 and HIV-2 CA proteins, we determined thermal stabilities of HIV-1 and HIV-2 CA NTD proteins at several NaCl concentrations by fluorescent-based thermal shift assays. Experimental data obtained showed that HIV-2 CA NTD was structurally more stable than HIV-1 CA NTD. Taken together, our results imply that distinct in vitro polymerization abilities of the two CA proteins are related to their structural instability/stability, which is one of the decisive factors for viral replication potential. In addition, our assay system described here may be potentially useful for searching for anti-CA antivirals against HIV-1 and HIV-2