The Host DHX9 DExH-Box Helicase Is Recruited to Chikungunya Virus Replication Complexes for Optimal Genomic RNA Translation.

Beyond their role in cellular RNA metabolism, DExD/H-box RNA helicases are hijacked by various RNA viruses in order to assist replication of the viral genome. Here, we identify the DExH-box RNA helicase 9 (DHX9) as a binding partner of chikungunya virus (CHIKV) nsP3 mainly interacting with the C-terminal hypervariable domain. We show that during early CHIKV infection, DHX9 is recruited to the plasma membrane, where it associates with replication complexes. At a later stage of infection, DHX9 is, however, degraded through a proteasome-dependent mechanism. Using silencing experiments, we demonstrate that while DHX9 negatively controls viral RNA synthesis, it is also required for optimal mature nonstructural protein translation. Altogether, this study identifies DHX9 as a novel cofactor for CHIKV replication in human cells that differently regulates the various steps of CHIKV life cycle and may therefore mediate a switch in RNA usage from translation to replication during the earliest steps of CHIKV replication.IMPORTANCE The reemergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In the absence of effective antiviral drugs, CHIKV infection has a significant impact on human health, with chronic arthritis being one of the most serious complications. The molecular understanding of host-virus interactions is a prerequisite to the development of targeted therapeutics capable to interrupt viral replication and transmission. Here, we identify the host cell DHX9 DExH-Box helicase as an essential cofactor for early CHIKV genome translation. We demonstrate that CHIKV nsP3 protein acts as a key factor for DHX9 recruitment to replication complexes. Finally, we establish that DHX9 behaves as a switch that regulates the progression of the viral cycle from translation to genome replication. This study might therefore have a significant impact on the development of antiviral strategies.

Peptides derived from evolutionarily conserved domains in Beclin-1 and Beclin-2 enhance the entry of lentiviral vectors into human cells.

Autophagy-related proteins such as Beclin-1 are involved in an array of complex processes, including antiviral responses, and may also modulate the efficiency of gene therapy viral vectors. The Tat-Beclin-1 (TB1) peptide has been reported as an autophagy-inducing factor inhibiting the replication of pathogens such as HIV, type 1 (HIV-1). However, autophagy-related proteins are also essential for the early steps of HIV-1 infection. Therefore, we examined the effects of the Beclin-1 evolutionarily conserved domain in TB1 on viral transduction and autophagy in single-round HIV infection or with nonreplicative HIV-1-derived lentiviral vectors. TB1 enhanced transduction with various pseudotypes but without inducing the autophagy process. TB1 augmented the transduction of human CD34+ hematopoietic stem/progenitor cells while maintaining their capacity to engraft in vivo into humanized mice. TB1 was as effective as other transduction additives and functioned by enhancing the adhesion and fusion of viral particles with target cells but not their aggregation. We also found that the N-terminal L1 loop was critical for TB1 transduction-enhancing activity. Interestingly, the Tat-Beclin-2 (TB2) peptide, derived from the human Beclin-2 protein, was even more potent than TB1 in promoting viral transduction and infection. Taken together, our findings suggest that the TB1 and TB2 peptides enhance the viral entry step. Tat-Beclin peptides therefore represent a new family of viral transduction enhancers for potential use in gene therapy.

Vpr expression abolishes the capacity of HIV-1 infected cells to repair uracilated DNA.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein binds to the cellular uracil-DNA glycosylase UNG2 and induces its degradation through the assembly with the DDB1-CUL4 ubiquitin ligase complex. This interaction counteracts the antiviral activity exerted by UNG2 on HIV-1 gene transcription, as previously reported by us. In this work, we show that Vpr expression in the context of HIV-1 infection markedly decreases UNG2 expression in transformed or primary CD4(+) T lymphocytes. We demonstrate for the first time that Vpr-UNG2 interaction significantly impairs the uracil excision activity of infected cells. The loss of uracil excision activity coincides with a significant accumulation of uracilated bases in the genome of infected cells without changes in cell division. Although UNG2 expression and uracil-DNA glycosylase activity are recovered after the peak of retroviral replication, the mutagenic effect of transient DNA uracilation in cycling cells should be taken into account. Therefore, the possible consequences of Vpr-mediated temporary depletion of endogenous nuclear UNG2 and subsequent alteration of the genomic integrity of infected cells need to be evaluated in the physiopathogenesis of HIV infection.

Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.

Human immunodeficiency virus type 1 (HIV-1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV-1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV-1-infected primary CD4(+) T-cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 2/3 of Tat are exported by HIV-1-infected cells during their lifespan. The function of extracellular Tat in HIV-1 infection might thus be more significant than earlier thought.

E5564 inhibits immunosuppressive cytokine IL-10 induction promoted by HIV-1 Tat protein.

BACKGROUND: In HIV-1 infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with progression of infection toward AIDS. HIV-1 Tat protein, by interacting with TLR4-MD2 at the membrane level, induces IL-10 production by primary human monocytes and macrophages. In the present study we evaluated the effect of the TLR4 antagonist Eritoran tetrasodium (E5564) on HIV-1 Tat-induced IL-10 production. FINDINGS: Here, we confirm that the recombinant HIV-1 Tat protein and the GST-Tat 1-45 fusion protein efficiently stimulate IL-10 production by primary monocytes and macrophages and that this stimulation is inhibited by blocking anti-TLR4 mAbs. We show that a similar inhibition is observed by preincubating the cells with the TLR4 antagonist E5564. CONCLUSION: This study provides compelling data showing for the first time that the TLR4 antagonist E5564 inhibits the immunosuppressive cytokine IL-10 production by primary human monocytes and macrophages incubated in the presence of HIV-1 Tat protein.

LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4+ T lymphocytes.

HIV-1 entry into CD4+ T lymphocytes relies on the viral and cellular membranes' fusion, leading to viral capsid delivery in the target cell cytoplasm. Atg8/LC3B conjugation to lipids, process named Atg8ylation mainly studied in the context of macroautophagy/autophagy, occurs transiently in the early stages of HIV-1 replication in CD4+ T lymphocytes. Despite numerous studies investigating the HIV-1-autophagy interplays, the Atg8ylation impact in these early stages of infection remains unknown. Here we found that HIV-1 exposure leads to the rapid LC3B enrichment toward the target cell plasma membrane, in close proximity with the incoming viral particles. Furthermore, we demonstrated that Atg8ylation is a key event facilitating HIV-1 entry in target CD4+ T cells. Interestingly, this effect is independent of canonical autophagy as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical step of its replication cycle.Abbreviations: BafA1: bafilomycin A1; BlaM: beta-lactamase; CD4+ TL: CD4+ T lymphocytes; PtdIns3K-BECN1 complex: BECN1-containing class III phosphatidylinositol 3-kinase complex; Env: HIV-1 envelope glycoproteins; HIV-1: type 1 human immunodeficiency virus; PM: plasma membrane; PtdIns3P: phosphatidylinositol-3-phosphate; VLP: virus-like particle.

HIV-1-associated PKA acts as a cofactor for genome reverse transcription.

BACKGROUND: Host cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle. RESULTS: NL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions. CONCLUSIONS: Altogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events.

Cellular kinases incorporated into HIV-1 particles: passive or active passengers?

Phosphorylation is one of the major mechanisms by which the activities of protein factors can be regulated. Such regulation impacts multiple key-functions of mammalian cells, including signal transduction, nucleo-cytoplasmic shuttling, macromolecular complexes assembly, DNA binding and regulation of enzymatic activities to name a few. To ensure their capacities to replicate and propagate efficiently in their hosts, viruses may rely on the phosphorylation of viral proteins to assist diverse steps of their life cycle. It has been known for several decades that particles from diverse virus families contain some protein kinase activity. While large DNA viruses generally encode for viral kinases, RNA viruses and more precisely retroviruses have acquired the capacity to hijack the signaling machinery of the host cell and to embark cellular kinases when budding. Such property was demonstrated for HIV-1 more than a decade ago. This review summarizes the knowledge acquired in the field of HIV-1-associated kinases and discusses their possible function in the retroviral life cycle.

Uracil DNA Glycosylase 2 negatively regulates HIV-1 LTR transcription.

Numerous cellular factors belonging to the DNA repair machineries, including RAD18, RAD52, XPB and XPD, have been described to counteract human immunodeficiency virus type 1 (HIV-1) replication. Recently, Uracil DNA glycosylase 2 (UNG2), a major determinant of the uracil base excision repair pathway, was shown to undergo rapid proteasome-dependent degradation following HIV-1 infection. However, the specific role of intracellular UNG2 depletion during the course of HIV-1 infection is not clearly understood. Our study shows for the first time that overexpression of UNG2 inhibits HIV-1 replication. We demonstrate that this viral inhibition is correlated with a marked decrease in transcription efficiency as shown by monitoring HIV-1 LTR promoter activity and quantification of HIV-1 RNA levels. Interestingly, UNG2 inhibits LTR activity when stimulated by Tat transactivator or TNFalpha, while barely affected using Phorbol ester activation. Mutational analysis of UNG2 indicates that antiviral activity may require the integrity of the UNG2 catalytic domain. Altogether, our data indicate that UNG2 is likely to represent a new host defense factor specifically counteracted by HIV-1 Vpr. The molecular mechanisms involved in the UNG2 antiviral activity still remain elusive but may rely on the sequestration of specific cellular factor(s) critical for viral transcription.

Coronavirus, the King Who Wanted More Than a Crown: From Common to the Highly Pathogenic SARS-CoV-2, Is the Key in the Accessory Genes?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that emerged in late 2019, is the etiologic agent of the current "coronavirus disease 2019" (COVID-19) pandemic, which has serious health implications and a significant global economic impact. Of the seven human coronaviruses, all of which have a zoonotic origin, the pandemic SARS-CoV-2, is the third emerging coronavirus, in the 21st century, highly pathogenic to the human population. Previous human coronavirus outbreaks (SARS-CoV-1 and MERS-CoV) have already provided several valuable information on some of the common molecular and cellular mechanisms of coronavirus infections as well as their origin. However, to meet the new challenge caused by the SARS-CoV-2, a detailed understanding of the biological specificities, as well as knowledge of the origin are crucial to provide information on viral pathogenicity, transmission and epidemiology, and to enable strategies for therapeutic interventions and drug discovery. Therefore, in this review, we summarize the current advances in SARS-CoV-2 knowledges, in light of pre-existing information of other recently emerging coronaviruses. We depict the specificity of the immune response of wild bats and discuss current knowledge of the genetic diversity of bat-hosted coronaviruses that promotes viral genome expansion (accessory gene acquisition). In addition, we describe the basic virology of coronaviruses with a special focus SARS-CoV-2. Finally, we highlight, in detail, the current knowledge of genes and accessory proteins which we postulate to be the major keys to promote virus adaptation to specific hosts (bat and human), to contribute to the suppression of immune responses, as well as to pathogenicity.

Retroviral Antisense Transcripts and Genes: 33 Years after First Predicted, a Silent Retroviral Revolution?

Paradigm shifts throughout the history of microbiology have typically been ignored, or met with skepticism and resistance, by the scientific community. This has been especially true in the field of virology, where the discovery of a "contagium vivum fluidum", or infectious fluid remaining after excluding bacteria by filtration, was initially ignored because it did not coincide with the established view of microorganisms. Subsequent studies on such infectious agents, eventually termed "viruses", were met with skepticism. However, after an abundance of proof accumulated, viruses were eventually acknowledged as defined microbiological entities. Next, the proposed role of viruses in oncogenesis in animals was disputed, as was the unique mechanism of genome replication by reverse transcription of RNA by the retroviruses. This same pattern of skepticism holds true for the prediction of the existence of retroviral "antisense" transcripts and genes. From the time of their discovery, it was thought that retroviruses encoded proteins on only one strand of proviral DNA. However, in 1988, it was predicted that human immunodeficiency virus type 1 (HIV-1), and other retroviruses, express an antisense protein encoded on the DNA strand opposite that encoding the known viral proteins. Confirmation came quickly with the characterization of the antisense protein, HBZ, of the human T-cell leukemia virus type 1 (HTLV-1), and the finding that both the protein and its antisense mRNA transcript play key roles in viral replication and pathogenesis. However, acceptance of the existence, and potential importance, of a corresponding antisense transcript and protein (ASP) in HIV-1 infection and pathogenesis has lagged, despite gradually accumulating theoretical and experimental evidence. The most striking theoretical evidence is the finding that asp is highly conserved in group M viruses and correlates exclusively with subtypes, or clades, responsible for the AIDS pandemic. This review outlines the history of the major shifts in thought pertaining to the nature and characteristics of viruses, and in particular retroviruses, and details the development of the hypothesis that retroviral antisense transcripts and genes exist. We conclude that there is a need to accelerate studies on ASP, and its transcript(s), with the view that both may be important, and overlooked, targets in anti-HIV therapeutic and vaccine strategies.

Electrostatic repulsion between HIV-1 capsid proteins modulates hexamer plasticity and in vitro assembly.

Capsid protein (CA) is the major component of the human immunodeficiency virus type 1 (HIV-1) core. Three major phosphorylation sites have been identified at positions S(109), S(149) and S(178) in the amino-acid sequence of CA. Here, we investigated the possible consequences of phosphorylation at these sites on the CA hexamer organization and plasticity using in silico approaches. The biological relevance of molecular modeling was then evaluated by analyzing the in vitro assembly properties of bacterially expressed CA bearing S(109)D, S(149)D, or S(178)D substitutions that mimic constitutive phosphorylation at these sites. We found that a constitutive negative charge at position 109 or 149 impaired the capacity of mature CA to assemble in vitro. In vivo, HIV-1 mutants bearing the corresponding mutation showed dramatic alterations of core morphology. At the level of CA hexamer, S(149) phosphorylation generates inter-monomer repulsions, while phosphorylation at position 109 resulted in cleavage of important bonds required for preserving the stability of the edifice. Addition of a negative charge at position 178 allowed efficient assembly of CA into core-like structures in vitro and in vivo and significantly increased CA hexamer stability when modeled in silico. All mutant viruses studied lacked infectivity since they were unable to produce proviral DNA. Altogether our data indicate that negative charges, that mimic phosphorylation, modulate assembling capacity of CA and affect structural properties of CA hexamers and of HIV-1 cores. In the context of the assembled core, phosphorylation at these sites may be considered as an event interfering with core organization and HIV-1 replicative cycle.

Antisense Transcripts and Antisense Protein: A New Perspective on Human Immunodeficiency Virus Type 1.

It was first predicted in 1988 that there may be an Open Reading Frame (ORF) on the negative strand of the Human Immunodeficiency Virus type 1 (HIV-1) genome that could encode a protein named AntiSense Protein (ASP). In spite of some controversy, reports began to emerge some years later describing the detection of HIV-1 antisense transcripts, the presence of ASP in transfected and infected cells, and the existence of an immune response targeting ASP. Recently, it was established that the asp gene is exclusively conserved within the pandemic group M of HIV-1. In this review, we summarize the latest findings on HIV-1 antisense transcripts and ASP, and we discuss their potential functions in HIV-1 infection together with the role played by antisense transcripts and ASPs in some other viruses. Finally, we suggest pathways raised by the study of antisense transcripts and ASPs that may warrant exploration in the future.

A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients.

The existence of an antisense Open Reading Frame (ORF) that encodes a putative AntiSense Protein (ASP) on the proviral genome of Human Immunodeficiency Virus type 1 (HIV-1) was a source of debate for 30 years. During the last years, some progresses have been made to characterize the cellular immune response against ASP in HIV-1 seropositive patients. However, no tools were available for the detection of antibodies to ASP in the plasma of HIV-1-infected patients during the natural course of the infection. The aim of our study was to develop a Luciferase Immuno-Precipitation System (LIPS) to monitor the quantitative detection of ASP-specific antibodies in the plasma of HIV-1-infected patients [antiretroviral therapy (ART) naive-patients, patients under ART and HIV-1 controllers], patients who discontinued antiretroviral drugs (ARV). We further used this approach to delineate the epitopes of ASP targeted by antibodies. Antibodies directed against ASP were detected in 3 out of 19 patients who discontinued ARV (15%) and in 1 out of 10 ART-naive patients (10%), but were neither detected in HIV-1 infected patients under ART nor in HIV-1 controllers. Individual variations in levels of ASP-specific antibodies were detected overtime. Both the conserved prolin-rich motif and the core 60-189 region of ASP were found to be essential for antibody recognition in the four patients tested positive for anti-ASP antibodies, who were all untreated at the time of sampling. Moreover, for two of these patients, increased levels of ASP-specific antibodies were observed concomitantly to viremia declines. Overall, our method may represent a useful tool to detect a humoral response to ASP in HIV-1-infected patients, which allowed us to confirm the expression of ASP during the course of HIV-1 infection. Further studies will be needed to fully characterize the humoral response to ASP in HIV-1-infected patients.

Is Uracil-DNA Glycosylase UNG2 a New Cellular Weapon Against HIV-1?

Uracil-DNA glycosylase-2 (UNG2) is a DNA repair protein that removes uracil from single and double-stranded DNA through a basic excision repair process. UNG2 is packaged into new virions by interaction with integrase (IN) and is needed during the early stages of the replication cycle. UNG2 appears to play both a positive and negative role during HIV-1 replication; UNG2 improves the fidelity of reverse transcription but the nuclear isoform of UNG2 participates in the degradation of cDNA and the persistence of the cellular genome by repairing its uracil mismatches. In addition, UNG2 is neutralized by Vpr, which redirects it to the proteasome for degradation, suggesting that UNG2 may be a new cellular restriction factor. So far, we have not understood why HIV-1 imports UNG2 via its IN and why it causes degradation of endogenous UNG2 by redirecting it to the proteasome via Vpr. In this review, we propose to discuss the ambiguous role of UNG2 during the HIV-1 replication cycle.

Insight into the mechanism of action of EP-39, a bevirimat derivative that inhibits HIV-1 maturation.

Maturation of human immunodeficiency virus type 1 (HIV-1) particles is a key step for viral infectivity. This process can be blocked using maturation inhibitors (MIs) that affect the cleavage of the capsid-spacer peptide 1 (CA-SP1) junction. Here, we investigated the mechanisms underlying the activity of EP-39, a bevirimat (BVM) derivative with better hydrosolubility. To this aim, we selected in vitro EP-39- and BVM-resistant mutants. We found that EP-39-resistant viruses have four mutations within the CA domain (CA-A194T, CA-T200N, CA-V230I, and CA-V230A) and one in the first residue of SP1 (SP1-A1V). We also identified six mutations that confer BVM resistance (CA-A194T, CA-L231F, CA-L231M, SP1-A1V, SP1-S5N and SP1-V7A). To characterize the EP-39 and BVM-resistant mutants, we studied EP-39 effects on mutant virus replication and performed a biochemical analysis with both MIs. We observed common and distinct characteristics, suggesting that, although EP-39 and BVM share the same chemical skeleton, they could interact in a different way with the Gag polyprotein precursor (Pr55Gag). Using an in silico approach, we observed that EP-39 and BVM present different predicted positions on the hexameric crystal structure of the CACTD-SP1 Gag fragment. To clearly understand the relationship between assembly and maturation, we investigated the impact of all identified mutations on virus assembly by expressing Pr55Gag mutants. Finally, using NMR, we have shown that the interaction of EP-39 with a peptide carrying the SP1-A1V mutation (CA-SP1(A1V)-NC) is almost suppressed in comparison with the wild type peptide. These results suggest that EP-39 and BVM could interact differently with the Pr55Gag lattice and that the mutation of the first SP1 residue induces a loss of interaction between Pr55Gag and EP-39.

Virus entry, assembly, budding, and membrane rafts.

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.

Refined study of the interaction between HIV-1 p6 late domain and ALIX.

The interaction between the HIV-1 p6 late budding domain and ALIX, a class E vacuolar protein sorting factor, was explored by using the yeast two-hybrid approach. We refined the ALIX binding site of p6 as being the leucine triplet repeat sequence (Lxx)4 (LYPLTSLRSLFG). Intriguingly, the deletion of the C-terminal proline-rich region of ALIX prevented detectable binding to p6. In contrast, a four-amino acid deletion in the central hinge region of p6 increased its association with ALIX as shown by its ability to bind to ALIX lacking the proline rich domain. Finally, by using a random screening approach, the minimal ALIX391-510 fragment was found to specifically interact with this p6 deletion mutant. A parallel analysis of ALIX binding to the late domain p9 from EIAV revealed that p6 and p9, which exhibit distinct ALIX binding motives, likely bind differently to ALIX. Altogether, our data support a model where the C-terminal proline-rich domain of ALIX allows the access of its binding site to p6 by alleviating a conformational constraint resulting from the presence of the central p6 hinge.

VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability.

BACKGROUND: The machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined. RESULTS: Here, we show that infectivity of HIV-1 mutants bearing S149A and S178A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S149A and S178A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and in vitro uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S149A and S178A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity. CONCLUSION: Defects generated at the level of core assembly and stability by S149A and S178A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.