Engineering human immunodeficiency virus 1 protease heterodimers as macromolecular inhibitors of viral maturation.

Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection.     

Inhibition of infectivity and replication of HIV-2 and SIV in helper T-cells by 2',3'-dideoxynucleosides in vitro

We have previously shown that 2',3'-dideoxynucleosides and their derivatives are potent inhibitors of the infectivity and cytopathic effect mediated by human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Recently a new group of retroviruses has been found in individuals originating from the western part of Africa. One member of this group is human immunodeficiency virus type-2 (HIV-2), previously designated lymphadenopathy-associated virus type-2 (LAV-2), found in patients with an AIDS-like illness. In this report, we show that 2',3'-dideoxynucleosides including 3'-azido-2',3'-dideoxythymidine (AZT) can significantly inhibit the infectivity and/or cytopathic effect of these retroviruses in vitro. Current data provide a rationale for considering 2',3'-dideoxynucleosides and their derivatives as experimental antiviral agents in individuals with illnesses caused by these new retroviruses.     

Spotlight on HIV-1 Nef: SERINC3 and SERINC5 Identified as Restriction Factors Antagonized by the Pathogenesis Factor

The Nef protein is an accessory gene product encoded by human immunodeficiency virus types 1 and 2 (HIV-1/-2) and simian immunodeficiency virus (SIV) that boosts virus replication in the infected host and accelerates disease progression. Unlike the HIV-1 accessory proteins Vif, Vpr and Vpu, Nef was, until recently, not known to antagonize the antiviral activity of a host cell restriction factor. Two recent reports now describe the host cell proteins serine incorporator 3 and 5 (SERINC3 and SERINC5) as potent inhibitors of HIV-1 particle infectivity and demonstrate that Nef counteracts these effects. These findings establish SERINC3/5 as restrictions to HIV replication in human cells and define a novel activity for the HIV pathogenesis factor Nef.     

Phosphorylation-dependent human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA.

In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.     

The human and simian immunodeficiency viruses HIV-1, HIV-2 and SIV interact with similar epitopes on their cellular receptor, the CD4 molecule

The cellular receptor for HIV-1 is the leucocyte differentiation antigen, CD4. Blocking of HIV-1 infectivity can be achieved with monoclonal antibodies (MAbs) to some, but not all epitopes of this antigen. We demonstrate here, by inhibition of virus infection, blocking of syncytium formation and inhibition of pseudotype infection with a panel of CD4 MAbs, that HIV-1, HIV-2 and simian immunodeficiency virus (SIV) isolates share the same cellular receptor, the CD4 glycoprotein. It is also shown that very similar epitopes of this molecule are involved in virus binding. We infer from these data that the binding sites on these viruses are highly conserved regions, and may therefore make good targets for potential vaccines. In addition, we show that cell surface expression of CD4 is similarly modulated after infection of cell lines by all the viruses.     

A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion

HIV-1 and other retroviruses occasionally undergo hypermutation, characterized by a high rate of G-to-A substitution. Recently, the human apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), first identified as CEM15, was shown to be packaged into retroviral virions and to deaminate deoxycytidine to deoxyuridine in newly synthesized viral minus-strand DNA, thereby inducing G-to-A hypermutation. This innate mechanism of resistance to retroviral infection is counteracted by the HIV-1 viral infectivity factor (Vif), which protects the virus by preventing the incorporation of APOBEC3G into virions by rapidly inducing its ubiquitination and proteasomal degradation. To gain insights into the mechanism by which Vif protects HIV-1 from APOBEC3G, we substituted several amino acids in human APOBEC3G with equivalent residues in simian APOBEC3Gs that are resistant to HIV-1 Vif and determined the effects of the mutations on HIV-1 replication in the presence and absence of Vif. We found that a single amino acid substitution mutant of human APOBEC3G (D128K) can interact with HIV-1 Vif but is not depleted from cells; thus, it inhibits HIV-1 replication in an HIV-1 Vif-resistant manner. Interestingly, rhesus macaque simian immunodeficiency virus 239 or HIV-2 Vif coexpression depleted the intracellular steady state levels of the D128K mutant and abrogated its antiviral activity, indicating that it can be a substrate for the proteasomal pathway. The HIV-1 Vif-resistant mutant APOBEC3G could provide a gene therapy approach to combat HIV-1 infection.     

Consistent and significant inhibition of human immunodeficiency virus type 1 envelope-mediated membrane fusion by beta-chemokines (RANTES) in primary human macrophages

Infection and entry of CD4(+) cells by human immunodeficiency virus type 1 (HIV-1) requires a coreceptor molecule, which, in concert with CD4, interacts with the viral envelope glycoprotein (Env), leading to membrane fusion. The principal coreceptors are the CCR5 and CXCR4 chemokine receptors. The suppressive effect of beta-chemokines, principally RANTES, on certain HIV-1 isolates was established before the discovery of the CCR5 receptor, and there have since been multiple reports confirming this initial observation. However, the inhibitory effect of beta-chemokines on HIV-1 infection of macrophages has been controversial. The current study focused on this issue in detail, with a reductionist approach, using assays that measure the effect of beta-chemokines solely on Env-mediated fusion. It is shown that under a variety of culture and differentiation conditions, RANTES maintains a significant and consistent inhibitory effect on CCR5-dependent Env-mediated fusion, and the role of these findings is discussed in relation to the role of beta-chemokines in HIV pathogenesis.     

Partial "repair" of defective NEF genes in a long-term nonprogressor with human immunodeficiency virus type 1 infection

A 36-bp deletion close to the 5' end of NEF that impaired Nef function was found in a long-term nonprogressor with human immunodeficiency virus type 1 (HIV-1) infection. Forms containing an adjacent duplication of 33 bp were also frequently observed. The duplication showed no homology to the deleted region but restored the overall length of the first variable loop of Nef. NEF alleles carrying the duplication were active in class I major histocompatibility complex (MHC-I) down-modulation and enhancement of virus infectivity. However, they showed little activity in CD4 down-regulation and were unable to stimulate viral replication in human peripheral blood mononuclear cells. Our study indicates that the enhancement of virion infectivity and the stimulation of HIV-1 replication in lymphocytes are distinct functions of Nef. Our findings also illustrate the capacity for repair of attenuating deletions in HIV-1 infection and suggest that a selective pressure for Nef-mediated MHC-I down-modulation and/or enhancement of virion infectivity exists.     

Virus attachment and replication are promoted after acquisition of host CD28 and CD152 by HIV-1

CD28 is constitutively expressed on CD4(+) cells, but its homologue CD152 is only weakly expressed after cell activation. To determine whether these 2 costimulatory molecules can be inserted into human immunodeficiency virus type 1 (HIV-1), virus was produced in CD28- and CD152-expressing Jurkat-derived cells. Both molecules were efficiently acquired by virions. Virus attachment and infectivity were more affected by CD152 than by CD28. Given that CD28/CD152-CD80/CD86 interactions play a dominant role in antigen presentation, it can thus be proposed that the association between virus-anchored host CD28/CD152 and cell-surface CD80/CD86 on target cells might have consequences for the transmission and pathogenesis of HIV-1.     

In vitro inhibition of HIV-1 infectivity by human salivas

Inhibitory factors to human immunodeficiency virus type 1 (HIV-1) in saliva may be responsible for the infrequent isolation of virus from saliva and also may account for the marked infrequency of salivary and/or oral transmission of HIV-1. Incubation of HIV-1 with human saliva followed by addition of the mixture to susceptible cells leads to partial or complete suppression of viral replication in vitro. We investigated the inhibitory effects of whole saliva and specific glandular salivas on HIV-1 infectivity as measured by viral-induced cytopathic effects in susceptible cells. Whole saliva contained marked inhibitory activity to HIV-1, strain HTLV-IIIB, and to virus infected cells. Submandibular saliva contained inhibitory activity, but of lesser quantity. Parotid saliva demonstrated no HIV-inhibitory activity. Whole saliva also appeared to contain filterable components that were inhibitory to lymphocyte growth. Passage through a .45 micron pore-size filter eliminated the viral inhibitory activity of submandibular saliva and some of the activity in whole saliva. All salivas except parotid incubated with HIV-1 followed by filtration were inhibitory suggesting that complexing of virus with high molecular weight, submandibular mucins may play a role in viral inhibition.     

Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody.

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.     

Role of protein N-glycosylation in pathogenesis of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1), the retrovirus responsible for acquired immunodeficiency syndrome (AIDS), contains two heavily glycosylated envelope proteins, gp120 and gp41, which mediate attachment of virions to glycosylated cell surface receptor molecules (CD4 antigens) and appear to be responsible for syncytium formation and associated cytopathic effects of this virus. A comprehensive study of the effects of N-linked glycoprotein processing inhibitors on HIV-1 replication, infectivity, cytopathicity, target-cell infectibility, syncytium formation, and gp120 electrophoretic mobility was conducted to assess the importance of protein glycosylation in the pathogenesis of HIV-1 in vitro. The electrophoretic mobility of gp120 was decreased when gp120 was synthesized in the presence of castanospermine or 1-deoxynojirimycin (inhibitors of glucosidase I), increased when gp120 was synthesized in the presence of 1-deoxymannojirimycin (mannosidase I) or swainsonine (mannosidase II), and unaffected when gp120 was synthesized in the presence of bromoconduritol (glucosidase II). Inhibition by tunicamycin (lipid-linked oligosaccharide precursor synthesis), castanospermine, 1-deoxynojirimycin, and 1-deoxymannojirimycin attenuated HIV-1 infectivity and blocked HIV-1-induced syncytium formation and cytopathicity, whereas bromoconduritol and swainsonine failed to have such effects. None of the inhibitors interfered with virus replication in acutely infected cells or affected the ability of target cells to form syncytia with untreated HIV-1-infected cells. These results demonstrate that protein N-glycosylation is critical to the pathogenesis of HIV-1 at the levels of viral infectivity and cytopathicity but not at the level of virus replication or of host-cell infectibility.     

Vpu: a multifunctional protein that enhances the pathogenesis of human immunodeficiency virus type 1

The Vpu protein is the smallest of the proteins encoded by human immunodeficiency virus type 1 (HIV-1). This transmembrane protein interacts with the CD4 molecule in the rough endoplasmic reticulum (RER), resulting in its degradation via the proteasome pathway. Vpu also has been shown to enhance virion release from infected cells. While much has been learned about the function of Vpu in cell culture systems, its exact role in HIV-1 pathogenesis is still unknown. This has been primarily due to the lack of a suitable primate model system since vpu is found only in HIV-1 and simian immunodeficiency viruses isolated from chimpanzees (SIVcpz), and three species of old world monkeys within the genus Cercopithecus. Several laboratories have developed pathogenic molecular clones of simian-human immunodeficiency virus (SHIV) in which the tat, rev, vpu and env genes of HIV-1 are expressed in the genetic background of SIV. The availability of such clones has allowed investigators to assess the role of Vpu in pathogenesis using a relevant animal model. This review will focus on the current understanding of the structure-function relationships of Vpu protein and recent advances using the SHIV model to assess the role of Vpu in HIV-1 pathogenesis.     

Expression of human CD4 in transgenic mice does not confer sensitivity to human immunodeficiency virus infection.

Transfection of the human CD4 molecule into mouse cells does not confer susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Expression of the human CD4 molecule in transgenic mice was seen to offer some new possibilities. However, transgenic mouse T cells expressing either the human CD4 receptor, or a hybrid human/mouse CD4 receptor alone or in conjunction with human major histocompatibility complex class I molecules, were refractory to in vitro HIV-1 infection. In addition, no infection was observed after in vivo HIV inoculation to mice of these various transgenic lines. Injection of recombinant gp160 viral protein to the transgenic mice did not alter their T and B cell populations. The existence of a dominant block in mouse cells that prevents HIV entry is discussed.